1769
Langmuir 1990,6, 1769-1773
Effect of the Addition of Ceramide to Dioleoylphosphatidylcholine Vesicles: An ESR and SEM Study Francesca Bonosi, Gabriella Gabrielli, Elena Margheri, and Giacomo Martini* Department of Chemistry, University of Florence, Via G . Capponi 9, 50121 Firenze, Italy Received February 27, 1990. In Final Form: June 1 , 19SO
Electron spin resonance spectroscopy of doxylstearic acids with the paramagnetic unit inserted in the 5,6,7,9,12, and 16 carbon position was used to analyze the dynamic and structural features of dioleoyl-
phosphatidylcholine (DOPC)vesicles in the presence and in the absence of ceramide (CER) and as a function of temperature. The sizes and the homogeneity of these lipid aggregates were checked by electron microscopy and turbidimetry. The order parameter, as calculated from the ESR line shapes, indicated that near room temperature the vesicles had a relatively high orientational organization from the polar surface down to the system of the double bonds of the oleoyl residues of DOPC, whereas the double-layer region beyond the double bond was increasingly fluid. At temperatures lower than the gel-liquid crystal phase transition of CER, T,, the presence of the sphingolipid did not alter significantly the motional properties of the hydrocarbon chains. At T 2 T,, the addition of CER affected only the lipid layer region from the surface down to the oleoyl double bonds with resulting increased fluidity. The double-bond region acted therefore as a boundary for the effect of CER. No definitive evidence of lipid interdigitationwas given by the results reported in this work.
Introduction Both vesicles and liposomes are typical curved bilayer systems, and they are accepted as possible models of biological membranes for their features, which quite well represent the real As curved bilayer systems, vesicles may be considered the closest unoriented lipid dispersions to be compared with the planar oriented bilayers and multibilayers. In the latter systems, the macroscopic anisotropy allows one to perform detailed studies on the angular dependency, which may be, in turn, timeaveraged by fast anisotropic motions in unoriented bilayers. Many experimental techniques have been used for studying curved bilayers, including spin resonance spectroscopies,4*5which are very suitable for obtaining detailed information about structural and dynamic properties of these systems. In this paper, we report a continuous-waveelectron spin resonance (ESR) and scanning electron microscopy (SEM) study on some dynamic and static properties of vesicles built up with a single phospholipid in the presence and in the absence of another membrane-typical lipid, a sphingolipid. In the vesicle preparation, we used dioleoylphosphatidylcholine (DOPC),which bears a double-bond system at C&lo in both fatty acid residues. This lipid is known to give easily curved bilayewe Thus it was possible to verify if the addition of compounds related to (1) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-lnter-
science: New York, 1982. (2) Strom-Jensen, P. R.; Magin, R. L.; D u n , F. Eiochim. Eiophys. Acta 1984, 769,179. (3) Maynard, V. M.; Magin, R. L.; Dunn, F. Chem. Phye. Lipids 1985, “9
.
a/, I.
(4) Silver, B. L. The Physical Chemistry of Membranes; Allen and Unwin: Boston, 1985. (5) Hemminga, M. A. Chem. Phya. Lipide 1983,32,323. (6) Koretanje, L. J.; van F a w n , E. E.; Levine, Y. K.Biochim. Biophys. Acta 1982, 982, 198.
biological components led to changes in some bilayer properties related to the nature of the lipid component. We used a simple ceramide from bovine brain (CER), without sugar residue and containing a mixture of fatty acids. The ceramide had the typical structure of the sphingolipids with two aliphatic chains, the first one being a carboxylic acid linked with an amidic bond to sphingosine, the second one being the sphingosine itself. The lack of the sugar residue allowed one to investigate the effect on liposomes of sugar-free precursors of glycolipids. In a previous work: it has been demonstrated that CER does not give black lipid membranes (BLMs) which are, together with vesicles, the most commonly accepted model for membranes. On turn, BLMs have been obtained when CER is mixed with the very mobile lipid monoolein, which bears a single, unsaturated chain. Thus, the main aim of the present paper was to verify if mixtures of CER with a vesicle-forming lipid really give closed vesicle bilayers and to investigate the molecular basis of this behavior. The analysis of the size of the ordered aggregates, as obtained from SEM and turbidimetry, and of their dynamics, as obtained from ESR line shapes, allowed us to ascertain the effects of the addition of the sphingolipid and of the presence of localized double bonds in the DOPC component.
Experimental Section Materials. The following products were used for the vesicle preparation:L-a-dioleoylphosphatidylcholine(DOPC, as a 20 mg/mL chloroform solution, Sigma Chemie, Darmstadt, FRG; MW 786.1, purity > 99%) and bovine brain ceramide (CER, Sigma Chemie, Darmstadt,FRG; averaged MW 608.8, purity 99 Yo ). n-Doxylstearic acids (n-DXSA,Molecular Probes, Eugene, OR, and Sigma Chemie) were used as received without fur-
-
~
~
~~
(7) Margheri, E.; Niccolai, A.; Gabrielli, G.; Ferroni, E. Colloids Surf.
In presa.
0743-7463/90/2406-1769$02.50/0@ 1990 American Chemical Society
Bonosi et al.
1770 Langmuir, Vol. 6, No. 12, 1990 ther purification. The formulas for the chemicals used are shown below:
0R1 =
Rz (CHz),CH=CH(CH~)$H3 DOPC
CH(OH)CH=CH(CH,),2CH, I CH -NHOCR I CHzOH CER
CHI(CHz)n~(CHz),,,COOH I
n=12 11 10 8 5
1
mDXSA m = 3 5-DXSA 4 BDXSA 5 7-DXSA 7 SDXSA 10 12-DXSA 14 lBDXSA
The bovine brain CER was a mixture of several products in which the fatty acid residue R was as follows: C16:0, 1.9%; C18:0, 32.3"; ; C20:4, 3.7%; C24:0, 10.9%; C24:1, 34.4%; 0thers, 16.8rcj(as determined by the manufacturer from gas chromatographic analysis). Vesicle Preparation. For the vesicle preparation, we used a procedure similar to that reported by Huang.* First, 1.5 mL of the chloroform solution of DOPC (20 mg/mL) was dried on the bottom of a vial under N1 flow. T o the resulting thin layer of lipid, 3 mL of phosphate buffer (Titrisol, Merck, Darmstadt, FRG pH 7) was added, and the aqueous dispersion of the lipid was sonicated at 333 K for 1h with the Branson Sonic B12 sonicator. A titanium microprobe and 50-60 W of power were used. The total lipid content was 10 mg/mL (1.27 mmol/L). CER-containing vesicles with the same total lipid content were prepared in the same manner. The DOPC/CER ratio was invariantly 1.5 w/w (1.16 mol/mol). The nitroxide insertion in the vesicle bilayers was achieved by adding very small amounts (less than 0.2% of the total liquid, in order to have DXSA/lipid ratio 1:200) of ethanol solutions of the appropriate DXSA after sonication took place. Although vesicles with different total lipid contents were also prepared, for most measurements we used the above samples which gave the most reproducible results. The sonication temperature of 333 K was chosen in order to be above the gel-liquid crystal phase transition temperatures of both DOPC (T, = 251 K)' and of CER (T, 323 K)$ we were therefore in fluidity conditions similar to those of natural membranes a t which DOPC layers may be curved. Methods. Scanning electron micrographs were obtained with the SEM-515 Philips system. The samples were prepared by evaporating a few drops of vesicle dispersion onto an aluminum plate. After drying, the samples were covered with a thin gold layer. Turbidimetry measurements were carried out at room temperature with UV-vis Lambda 5 Perkin-Elmer spectrometer. ESR spectra were taken with the aid of a Bruker Model 200D spectrometer operating at X-band (-9.5 GHz) and equipped with t h e Aspect 2000 data-handling system for spectral accumulation and magnetic parameter calculation. Microwave power and signal modulation were such as to avoid signal saturation and overmodulation. Temperature was varied with the Bruker ST100/700 variable-temperature accessory.
-
(8) Huang, C. Biochemistry 1969, 8,344. (9) Fidelio, C. D.; Maggio, B.; Cumar, F. A. Biochim. Biophys. Acta 1986,854,231.
Langmuir, Vol. 6, No. 12, 1990 1771
Addition of Ceramide to DOPC Vesicles
qk
7-DXSAA
&XS
B
Figure 2. Scanning electron micrographs of D O P C / 6 D X S A vesicles (a) and DOPC/CER/5-DXSA vesicles (b):total lipid content, 10 mg/mL; total lipid/nitroxide ratio > 200.
SA
n" nv
A
16- DX S A
Figure 4. ESR spectra at 293 K of DOPC vesicles containing stearic acid nitroxides (lipid/nitroxideratio > 200). carried out on this system under addition of hypertonic salt solution. These did not give evidence of changes of volume and absorbance, which was a further proof against vesicle formation. The failure of osmotic shock is indeed a definitive proof of absence of closed curved bila~ers.'~J~ Details of the properties of the vesicle at a molecular level were obtained from ESR spectra of n-DXSA inserted as molecular probes in the organized assemblies. Figure 4 reports the ESR absorptions at 293 K due to 5-, 7-, 9-, 12-, and 16-DXSAin DOPC vesicles. The 5- and 7-DXSA spin labels reported the dynamics near the surface of the bilayers whereas 12- and 1bDXSA acted as probes of the more hydrophobic portion of the bilayer. Exactly the same line shapes were obtained at 293 K when the same probes were inserted in DOPC/CER vesicles. The ESR line shapes of Figure 4 reflected the motional properties of the environment in the proximity of the unpaired electron. In curved bilayer systems, the motion of the hydrocarbon .~ chains may be assumed as a fast anisotropic m ~ t i o nTo allow for the evaluation of the motional anisotropy of the fatty acid residues, the concept of "order parameters" was introduced'e-20 as derived from the model proposed by Saupe:21s22
Figure 3. Scanning electron micrograph of M aqueous dispersion of CER (20 mg of CER in 3 mL of phosphate buffer).
Si = (1/2)(3 COS' Bi - 1) (2) where Bi is the angle between the x , y, and z axes and the
whereas it entered within bilayered phospholipids when in a concentration lower than 30-40s mol/mol. Previous work carried out in this laboratory' has demonstrated that the same ceramide used here gives wellcharacterized spreading isotherms which indicate the presence of a liquid-condensed phase. The CER alkyl chains strongly interact with an almost vertical arrangement with respect to the surface. The mean value of the critical packing, as calculated according to the procedure reported by Maggio,13 was 1.19 this value rules out the vesicle formation. Additional turbidimetry runs were
(13) Msggio, B. Biochim. Biophys. Acto 1986,815,245. (14) Bengham, A. D.; Degier, J.; Greville, G. D. Chem. Phya. Lipids 1967, 1 , 225. (15) Kano. K.; Romero, A,; Djermouni, B.; Ache, H. J.; Fendler, J. H. J. Am. Chem. Soc. 1979,101,4030. (16) Hubbel, W.L.;M&nnell, H. M. J.Am. Chem.Soc. 1971,93,314. (17) Seelip. J. J . Am. Chem.Soe. 1910.92, 3881. (181 Griffith. 0. H.; Jost, P. C. In Spin Labeling. Theory and Applications; Berliner, L. J., Ed.;Academic Preas: New York, 1976; Vol. 1, pp 453-523. (191 Gaffney,B. J. ref 18, pp 567-571. (20) Marsh, D. In Membrane Spectroscopy; GreU, E.,Ed.; Springer Verlsg: New York, 1981; pp 51-142 and references therein. (21) Saupe, A. 2.Naturforsch. 1964.19A. 161. (22) Saupe, A. Angew. Chem., Znt. Ed. Engl. 1968, 7,W.
1772 Langmuir, Vol. 6, No. 12, 1990
Bonosi et al.
33.00
273 K
0.80
28.00 3
\
E
c
0
(3
I
\
0.40
\
a" 23.00 0.00
1 4
18.00 . 213
a
9
14
Doxyl position I
263 Temperature
I
313
I
363
(K) 0.40-
Figure 5. Experimental separation between outer peaks ( A ! )
as a function of temperature of doxyl nitroxides inserted in DOPC vesicles: (A) DOPC/B-DXSA; (0) DOPC/CER/S-DXSA; (0) DOPC/G-DXSA; (*) DOPC/CER/G-DXSA; (A) DOPCI7DXSA; ( 0 )DOPC/CER/7-DXSA.
E
director perpendicular to the lipid bilayer. Assuming fast anisotropic motion in unoriented lipid bilayer and multibilayer systems, the ESR line shapes reflect the timeaveraged positions of the spin labels;20i.e., Si in eq 2 arises from a time averaging. From the experimental peak separations, All and A l are determined, which give4J7
v)
0.20-
0.00
4 6 0 S,= ([All-A,l/IA,,Doxyl position (Axx + ~ y y ~ / ~ l ~ (3)~ o ~ c ~ ~ ~ ~ ~ / ~ o ~ ~ ~ ~ ~ Figure. 6. Order parameter S d at 273 and at 363 K as a function where ao(cryst)/ao(bil) is the correction which takes into of the position of the doxyl group on the alkyl chain of stearic parameter account the polarity of the medium. The Smol DOPC/n-DXSA; (*) DOPC/CER/n-DXSA. acid (0) is defined for the long molecular axis on the DXSA spin probes. Calculation of Smo1 was then reduced to measuring of the doxyl group on the alkyl chain of stearic acid for the experimental separation of inner and outer spectral both DOPC and DOPC/CER vesicles. The shape of the splitting as it is shown in Figure 4. Figure 5 shows the order parameter prpfile looked very similar to that in experimental separations (in G) of 5-, 6-, and 7-DXSA in general observed by NMR for fatty acids of a membrane DOPC and DOPC/CER as a function of temperature. The lipid. The orientational order was relatively high from the three nitroxides gave All changes which were very similar water-lipid interface down to the inner side of the double for temperatures lower than 273 K, whereas in the range layer, at least down to the region sensed by the 12-DXSA 273-323 K the disorder of the nitroxide long chain was probe, that is, in the proximity of the double-bond system greater the farther the nitroxide moiety was from the biof DOPC. In the hydrophobic layer region far enough from layer surface. A t temperatures higher than 320-330 K the double bonds sensed by 16-DXSA, the intermolecu(which approximately corresponded to the gel-liquid lar organization was largely disrupted, and the probe had crystal phase transition of CER), the effect of added CER an increasingly smaller anisotropic motion. We must, became apparent and was manifested by an abrupt however, clearly point out that the value of Smo1 0.2 for crossover of the graphed curves. An important question 16-DXSA should be regarded with some caution because arises at this point. It is known that glycolipids and phos€or low values of Smolthe oriented spectra will tend to the pholipids show peculiar solubility behavior as recently isotropic three-line nitroxide spectra (Sd 0). Computed reviewed by C ~ r a t o l o .Published ~~ data on the mutual spectra could be used24-26to obtain more reliable values miscibility of phospholipids and gluco- or galactocereof the order parameters. However, the observed line shapes brosides below the gel-liquid crystal phase transition were highly representative of extremely disordered systems. temperature indicate the coexistence of a phospholipidIn no case did the addition of CER seem to alter the order rich fluid phase and a glycolipid-rich rigid phase. Since of the bilayers. As expected from the All behavior, this free fatty acids are expected to preferentially spend their variation occurred at temperatures higher than T,of CER. time in the fluid phase, the region sensed at low temperFigure 6 reports the Smol values at 363 K for 5-, 6-, 7-, ature by the fatty acid spin probes used in this work should and 9-DXSA inserted in DOPC and DOPC/CER. Since be the fluid DOPC domains and not the rigid CER 9-DXSA probed the layer region immediately above the domains. Although CER is a sugar-free sphingolipid, the above considerations might explain the lack of a strong (24) Van, 5.P.; Birrel, G . B.; Griffith, 0. H. J . Magn. Reson. 1974,15, effect of CER below 320 K, whereas at 7' > 320 K a true 444. (25) Libertini, L. J.; Burke, C. A.; Joet, P. C.; Griffith, 0. H. J . Magn. mixing seemed to occur. Figure 6 reports the Smol values Reson. 1974,15,460. at 273 K calculated with eq 3 as a function of the position (28) Schneider, D. J.; Freed, J. H. In Biological Magnetic Resonance. Smol
N
-
~
(23) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 111.
~~
-
~
Spin Labeling. Theory and Applications; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1989 Vol. 8,pp 1-76.
Addition of Ceramide to DOPC Vesicles double bonds of the oleoyl residues of DOPC, the results clearly indicated that a t high temperature the addition of CER induced higher disorder, that is, a higher fluidity,from the interface down to the double-bond limit. In fact, almost t h e same order was reported by 9-DXSA independently of the presence of CER, and this was further proved by the lack of differences in the spectral shapes of 12- and 16-DXSA, which were more or less buried beyond the oleoyl double bonds. Both probes showed very low order degrees (&,,I 0.2) independently of the presence of CER. Cestaro et al.27 have studied the dynamics of egg phosphatidylcholine vesicles containing ceramide galactose-3-sulfate. This is a sphingolipid with a galactose-3-sulfate polar group and hydrophobic chains of different composition. The authors find that the sulfatide inserted in the vesicles reduces the fluidity of hydrocarbon chains both near the bilayer surface (by using 5-DXSA as a probe) and, although in a much lesser extent, in the hydrophobic region (by using 16-DXSA). However, the result of Cestaro et al. and the results of the present work cannot be compared since different systems were used. First, the egg PC is not a simple phospholipid as DOPC is, but it contains a mixture of fatty acids. Second and more importantly, the above authors used a charged ceramide with a sulfocarbohydrate group protruding from the membrane surface. This is able to give a variety of interactions,28including electrostatic interactions which are expected to reduce the system fluidity. The CER we used, beside the 18-carbon chain of the sphingosine which extends down in the membrane almost parallel to the oleoyl residues of DOPC, had fatty acid chains whose length ranged from 16 to 24 carbon atoms. In such cases, the concept of “interdigitation” has arisen, that is, the possibility of an extension of longer alkyl chains of a monolayer among the shorter chains of the opposite monolayer of the bilayer.29*30This question has been elegantly approached by Grant and ~0-workers3~~3~ through the use of synthesized long-chain (C24) and short-chain (CIS)
Langmuir, Vol. 6, No. 12, 1990 1773 spin labeled glycoceramides. On the basis of the order parameter S, the authors show that in liposomes built with several phospholipids, including DMPC and DPPC, the interdigitation occurs with the terminal part of the long fatty acids crossing the membrane hydrophobic center. The spin labels we used in this work had the same 18-carbon length as DOPC. The finding of almost equivalent order parameter in the presence or in the absence of CER seemed to rule out interdigitation from long-chain residues of CER in the bilayer region very near the (3-16 position sensed by 16-DXSA. This suggested that the terminal part of the long chains of CER was folded back upon itself to form a focus of disruption. However, this sketch cannot be considered entirely unambiguous, and more experimental data are necessary to confirm or reject it.
Conclusions The use of SEM and turbidimetry allowed us to establish that DOPC gave vesicles which were largely multilamellar (from turbidimetric measurements), and this structure was maintained after insertion into the organized assemblies of paramagnetic probes with a stearic hydrocarbon chain. The sphingolipid CER could be added to DOPC vesicles with a resultant increase of their sizes, whereas CER was unable to give true vesicles. From the analysis of the ESR line shapes of the doxy1 nitroxides inserted in the curved bilayers, a significant orientational order was observed around room temperature from the bilayer polar surface down to the double-bond system of the oleoyl residues, whereas the bilayer structure became suddenly fluid well below this double-bond system. At a temperature higher than T,(CER), no dependence on CER presence was observed in the fluidity of the inner hydrophobic region. CER acted as an effective fluidizing agent above the double-bond region where the orientational order was appreciably decreased by the presence of the sphingolipid. No definitive evidence was obtained of interdigitation of the long fatty acid residues of CER.
(27) Cestaro, B.; Cervato, G.; Marcheeini, S.; Viani, P.; Pistolesi, E.; Oliva, C. Chem. Phys. Lipids 1983,33, 251. (28) Farooqui, A. A,; Rebel, G.; Mandel, P. Life Sci. 1977, 20, 5694. (29) Davis, P. J.; Keough, K. M. W. Biophys,.J. 1985,48, 915. (30) Huang, C.; Mason, J. T. Biochim. Biophys. Acta 1986,864,423
Acknowledgment. Thanks are due to Minister0 della Pubblica Istruzione (MPI) and to Consiglio Nazionale delle Ricerche (CNR) for financial support. F. B. also thanks ENI-Ricerche for a fellowship. Registry No. DOPC, 4235-95-4.
and references therein. (31) Grant, C. W. M.; Mehlhorn, I. E.; Florio, E.; Barber, K. R. Biochim. Biophys. Acta 1987,902,169.
(32) Mehlhorn, I. E.; Florio, E.; Barber, K. R.; Lordo, C.; Grant, C. W. M. Biochim. Biophys. Acta 1988, 939, 151.
