3161
Langmuir 1996,11, 3161-3166
Intermolecular Interactions of Lithocholic Acid Derivatives with Phospholipid as Bilayer Membrane Zi-Chen Li,? Fu-Mian Li,? Shinya Arase, Shinji Takeoka, and Eishun Tsuchicla" Department of Polymer Chemistry, Waseda University, Tokyo 169, Japan Received November 15, 1994. In Final Form: May 4, 1995@ Three single chain lithocholic acid derivatives with a steroidal moiety in the center of the hydrocarbon chain and two head groups at both ends of the chain were synthesized and characterized. Due to their high compatibility with phospholipids, they can be incorporated homogeneously into the phospholipid bilayer membrane. The resulting mixed vesicles showed high stability against aggregation by monitoring the turbidity change in the vesicle suspensions. The entire membrane-spanning packing thus locates the steroidal moiety at the center of the bilayer. The interaction of the steroidal moiety with lipid molecules at the center ofthe bilayer resulted in higher mobility of lipid molecules below the Tcof the host phospholipid membrane as clarified by temperature-dependent 'H NMR, fluorescence depolarization anisotropy, and differential scanning calorimetry (DSC). The results were compared with those of cholesterol, whose steroidal moiety is located near the surface of the membrane.
Introduction
vesicles.s-ll Here, we reported the synthesis of three lithocholic acid derivatives (1,2,3) with different hydroThe effect of cholesterol on the packing states of phobic chain lengths. The chain lengths of these three phospholipids in a bilayer membrane has been the subject molecules were carefully designed so that 2 and 3 in the of many research studies due to the significant role of membrane-spanning packing state were almost the same cholesterol in the stability of natural cell membranes.'-3 length as the thickness of the gel state bilayer of dipalmitoylphosphatidylcholine (DPPC) and dimyrisCholesterol enhances the molecular packing of the bilayer toylphosphatidylcholine (DMPC),respectively. 1 is longer membrane in a liquid crystalline state, resulting in higher than both the DPPC and DMPC bilayers. The effects of stability of the dispersion state. Below the gel-to-liquid the steroidal moiety a t the center of the bilayer on the crystalline phase transition temperature of the host packing state of host phospholipid membrane, and the phospholipid membrane, cholesterol affords flexibility to relationship between these effects and the chain length the membrane. The rigid steroid ring of cholesterol, of the membrane-spanning lipids, together with the located near the surface of the membrane and oriented stability of the mixed vesicles, were studied by temperparallel to the hydrophobic chains of the phospholipids, ature-dependent 'H-NMR spectra, fluorescence depolaris. considered to play a n important role in such unique properties of the mixed membrane with c h o l e ~ t e r o l . ~ ~ ~ization anisotropy, and differential scanning calorimetry (DSC). Some efforts have been made in recent years to introduce a steroidal moiety as a structural component of synthetic lipids. The phospholipid derivatives having the steroidal moieties so as to be located near the surface of the membrane decreased the curvature of the vesicles and afforded the high stability of the membrane.'jv7 In order to clarify the different effects of the location of a rigid steroidal moiety at the center of the bilayer membrane, lithocholic acid was used as a rigid segment of a membrane-spanning lipid, having two head groups a t both ends of the long hydrophobic chain. This idea is based on the results that membrane-spanning lipids, either with a macrocyclic structure or a rigid moiety a t the center of a hydrophobic chain, can be arranged in the entire membrane-spanning packing state within the bilayer and can increase the thermal stability ofthe mixed
* To whom all correspondence should be addressed: Tel, +8133209-8895;f a , +813-3209-5522. t Present address: Department of Chemistry, Peking University, Beijing 100871, China. Abstract published in Advance ACS Abstracts, July 1, 1995. (1)Finegold L. Cholesterol in Membrane Models; CRC Press: Boca Raton, FL, 1993. (2)Yeagle, P. L. Biochim. Biophys. Acta 1985,822,267. (3)Keough, K.M. W.; Giffin, B.; Matthews, P. L. J.Biochim. Biophys. Acta 1989,983,51. (4)Stockton, G. W.; Smith, I. C. P. Chem.Phys.Lipids 1976,17,251. (5)Tilcock, C.P.S.;Bally, M. B.; Farren, S. B.; Cullis, P. R.; Grunner, S. M. Biochemistry 1984,13,2696. (6)Groves, J. T.;Neumann, R. J . Am. Chem. SOC.1989,111,2900. (7)Tsuchida, E.;Komatsu, T.; Babe, T.; Nakata, T.; Nishide, H.; Inoue, H. Bull. Chem. SOC.Jpn. 1990,63,2323. @
Experimental Section Materials. All reagents and chemicals were obtained from commercial sources and used without further purification. 11Bromo-1-undecanol,8-bromo-1-octanol,and lithocholic acid (3ahydroxy-5/3-cholan-24-oic acid)were purchased from Tokyo Kasei Co. 6-Bromo-1-hexanolwas from Aldrich Chemical Co. DPPC and DMPC were from Sigma Chemical Co. Cholesterol was from Kanto Chemical Co. and was recrystallized from methanol before use. 1,6-Diphenyl-1,3,5-hexatriene (DPH)was from Tokyo Kasei Co. and was used as a 1mM THF solution. All the solventsused were dried and purified by conventional methods before use. Characterization. Infrared spectra were recorded on a JASCO FT/IR-5300 spectrometer. lH-NMR spectra were recorded on a JEOL GSX-400 or JEOL GXS-270 instrument. Chemical shifts are expressed in ppm downfield from Mersi. FAB-MS spectra were measured with a JEOL DX-303 spectrometer. Thin-layerchromatography(TLC)was carried out on 0.2-mm precoated plates of silica gel 60 F-254 (Merk). Purification was performed by silica gel 60 (70-230 mesh, Merk). Synthesis of the Three Lithocholic Acid Derivatives. The four-step synthesis ofthe lithocholic acid derivatives is given
in Scheme 1. 3a-((Hydroxysuccinyl)oxy)-S/3-cholanicAcid (A). Lithocholic acid (3.76 g, 10 mmol) and succinic anhydride (1.2 g, 12 "01) were refluxed in 60 mLof dry toluene for 10h. On standing at room temperature, A was crystallized from the solution and (8)Fuhrhop, J.-H.; David, H.-H.; Mathieu, J.; Liman, U.;Winter, H.-J.; Boekman, E. J . Am. Chem. SOC.1986,108,1785. (9)Yamauchi, K.; Moriya, A.; Yamada, K.; Hosokawa, T.; Higuohi, T.; Kinoshita, M. J . Am. Chem. SOC.1990,112,3188. (10)Moss, R. A,; Li, J.-M. J . Am. Chem. SOC.1992,114,9227. (11)Menger,F.M.;Littau, C.A. J.Am. Chem.Soc. 1993,115,10083.
0743-746319512411-3161$09.00/00 1995 American Chemical Society
Li et al.
3162 Langmuir, Vol. 11, No. 8, 1995 Scheme 1. Synthetic Route of the Three Lithocholic Acid Derivatives
Lithocholic acid
SOCl,
w
reflux, 4hrs
In)
Cl!CH2CH2
6
HO(CH2),Br
benzene, room t h p . (6)
then was recrystallized twice from methanol: yield 4.0 g (84%); white powder, mp 238.7 "C;Rf= 0.37 (silica gel; l O / l , chloroform/ methanol). IR (KBr): 3500-2500 cm-l (-OH, acid), 1709 cm-l (carbonyl,acid), 1732 cm-l (carbonyl,ester);'H NMR (400 MHz, CDC13/CD30D, 80/20): 6 0.64 (s, 3H, CISsteroid), 0.91 (s, 6H, C19, C Zsteroid), ~ 1.5-1.6 (m, 2H, CZp, C15p steroid), 1.6-1.9 (m, 5H, Cla, C4a, C&3,C16a, C22-1H steroid), 1.96(d, lH, C1$ steroid), 2.2-2.4 (m, 2H, c23 steroid), 2.6 (m, 4H, -O(C=O)CH2CH2(O=C)O-), 4.8 (m, lH, C3 steroid), 0.94-1.5 (m, 18H,all others). 3a-((Chlorosuccinyl)oxy)-5/3-cholanic Acid Chloride (B). A (0.952 g, 2 mmol) and freshly distilled SOClz (2.38g, 20 mmol) were mixed and a drop of dry DMF was added as catalyst. The mixture was then refluxed for 4 h. After removal of the excess SOC12, the residue was dried completely in uucuo. It was used directly for the followingreaction without purification. Estimated yield: 90%. IR(neat): 1797cm-l (carboxylicacid chloride),1732 cm-l (carbonyl, ester). 3a411'-(((Bromoundecyl)oxy)succinyl)oxy)-24-( 11'-bromoundecyl)-5B-cholanate(C). 11-Bromoundecanol (1.05 g, 4.2 mmol) and (dimethy1amino)pyridine (0.488 g, 4 mmol) were dissolved in 20 mL of dry benzene. To this solution was added the above-obtained B dissolved in the same solvent with stirring over 20 min. The reaction proceeded overnight at room temperature. After removal of the solvent, purification of C was achieved by silica gel column chromatography with chloroform as solvent: viscous liquid; yield 0.85 g (45%);R f = 0.4 (silica gel; 15011, chlorofordmethanol). IR (neat): 1734 cm-l (carbonyl, ester), 723 cm-' (-CH2-). n > 4,646,563 cm-l (C-Br); 'H-NMR (400 MHz, CDC13): 6 0.64 ( 6 , 3H, CISsteroid), 0.91 (s, 6H, C19, CZIsteroid),2.2-2.4 (m, 2H, c23 steroid), 2.6 (m, 4H, -O(C-0)CHzCHz(O=C)O-), 3.5 (t, 4H, Br-CHZ-), 4.1 (t, 4H, -CH20(C=O)-), 4.8 (m, lH, C3 steroid),0.94-1.96 (m, 62H, all others).
(C-Br); 'H-NMR (270 MHz, CDC13): 6 0.64 (s, 3H, CISsteroid), 0.91 (s, 6H, C19, CZIsteroid), 2.2-2.4 (m, 2H, c23 steroid),2.6 (m, 4H, -O(C=O)CHzCH2(0=C)O-), 3.4 (t, 4H, Br-CH2-), 4.1 (t, 4H, -CH20(C=O)-), 4.8 (m, lH, C3 steroid), 0.94-1.98 (m,50H, all others). 3a-((((B'-Bromohexyl)oxy)succinyl~oxy)-24-(6'-bromohexyl)-5/3-cholanate(E). From B and 6-bromohexanol, the followingresults were obtained: viscous liquid; yield 0.48 g (30%); Rf=0.46 (silica gel; 100/1,chlorofodmethanol). IR (neat)1734 cm-l (carbonyl, ester), 729 cm-l (-CH2-ln n > 4,646, 561 cm-l (C-Br); 'H-NMR (270 MHz, CDC13): 6 0.64 (s,3H, CISsteroid), 0.91 (s,6H,CIS,C Zsteroid), ~ 2.2-2.4 (m, 2H, c23 steroid),2.6 (m, 4H, -O(C=O)CH2CH2(0=C)O-), 3.4 (t, 4H, Br-CH2-), 4.1 (t, 4H, -CHzO(C=O)-), 4.8 (m, lH, C3 steroid), 0.94-1.98 (m,42H, all others). 3a-((((11'-(Trimethy1amino)undecyl)oxy)succiny1)oxy)2441l'-(trimethylamino)udecyl)-5~-cholanate Dibromide (1). C was quaternized with trimethylamine (TMA)in dry DMF at 60 "Cfor 72 h. DMF was removed in uucuo, and the residue was further purified by silica gel column chromatography with chloroform and methanol as solvent (gradually changing ratio): waxy solid, yield 0.67 g (70%), Rf = 0.52 (silica gel; 65/30/5, chlorofodmethanoYwater). IR (neat): 1734 cm-l (carbonyl, ester), 723 cm-l (-CHz-). n > 4; 'H-NMR (270 MHz, CD3OD): 6 0.64 (s, 3H, CISsteroid),0.91 (s,6H, CIS,Czl steroid),2.2-2.4 (m, 2H, c 2 3 steroid), 2.6 (m, 4H, -O(C=O)CH&H2(O==C)O-), 3.2 (9, 18H, -N(CH3)3), 3.4(t, 4H, -CHzN-), 4.1 (t,4H, -CH20(C=O)-), 4.8 (m, lH, C3 steroid),0.94-1.96 (m, 62H, all others).
3a-((((8'-Bromooctyl)oxy)succinyl)oxy)-24(8'-bromooctyU-5fl-cholanate (D). From B and 8-bromooctanol, the fol-
g (60%);Rf=0.50 (silica gel;65/30/5,chlorofodmethanolater). IR (neat): 1734 cm-l (carbonyl, ester), 725 cm-l (-CHz-). n > 4; 'H-NMR (270 MHz, CD30D): 6 0.64 (s,3H, Cle steroid),0.91 (s, 6H, (219,Czl steroid),2.2-2.4 (m, 2H, c23 steroid),2.6 (m,4H, -O(CPO)CH2CH2(0=C)O-), 3.2 (s,18H, -N(CH3)3), 3.4 (t,4H,
lowing results were obtained: viscous liquid; yield 0.6 g (35%); R f = 0.23 (silica gel; 6/1, hexane/acetyl acetate). IR (neat): 1736 cm-l (carbonyl, ester), 725 cm-l (-CHZ-)~n > 4,644,565 cm-l
3a-((((8'-(trimethylamino)octyl)oxy)succinyl)oxy)-24(8'(trimethylamino)octyl)-5/3-cholanate Dibromide (2). From D and TMA, the following were obtained: waxy solid, yield 0.4
Langmuir, Vol. 11, No. 8, 1995 3163
Lithocholic Acid Derivatives -CH2N-), 4.1 (t,4H, -CH20(C=O)-), 4.8 (m, lH, C3 steroid), 0.94-1.96 (m, 50H, all others). FAB-MS (M - Br)+= 896,898. 3a-(( ( ( 6 -(trimethylamino)hexy1)oxy) succiny1)oxy)-24(6-(trimethylamino)hexyl)-S/3-cholanateDibromide (3). From E and TMA, the followingwere obtained: waxy solid,yield 0.36 g (60%);Rf = 0.50 (silica gel; 65/30/5, chlorofodmethanoll water). IR (neat): 1734cm-l (carbonyl,ester), 723 cm-l (-CH2n > 4; lH-NMR (270 MHz, CD30D): 6 0.64 (s,3H, CISsteroid), 0.91 (s, 6H, C19, C21 steroid), 2.2-2.4 (m, 2H, c23steroid), 2.6 (m, 4H, -O(C-O)CH2CH2(0=C)O-), 3.2 (s, 18H, -N(CH3)3), 3.4 (t, 4H, -CHzN-), 4.1 (t,4H, -CHzO(C-O)-), 4.8(m, lH, C~steroid), 1.96-0.94 (m, 42H, all others). FAB-MS (M - Br)+= 842,844. Turbidity Change Measurement. Small unilamellar vesicles were prepared by sonication (probe type, 60 W, N2, 55 "C, 10 min, [lipid]= 5 mM) of the CH3OH-cast films of DPPC (or with cholesterol) and the required molar ratio of 2 in deionized water. The absorbance was measured at 400 nm on a Shimadzu MPS-2000 spectrophotometer at room temperature; then the samples were incubated at 4 "C, and the relative absorbance change at 400 nm was measured with time. Temperature-Dependent 'H-NMR. Vesicles were prepared by sonication (probe-type,60 W, Nz, 50 "C, 10 min) of CH30Hcast films of DPPC (or with cholesterol) and the required molar ratios of 1,2, or 3 in D2O. The total lipid concentration was 20 mM. The vesicles were filtered through a 0.45-pm cellulose acetate filter (Advantec,Toyo Roshi Co.) and incubated at room temperature for 2 h. lH NMR spectra were recorded on a JEOL FX-9OQ Fourier transform spectrometer. The HDO signal (4.8 ppm) was used as an external reference. Fluorescence Depolarization Anisotropy of the Mixed Vesicles. Small unilamellar vesicleswere prepared in the same way as the turbidity change measurement with a total lipid concentration of 1mM (55 "C for DPPC, 35 "C for DMPC). To 10 mL of the above prepared vesicles filtered through a 0.45-pm filter, 10pL of 1mM DPH in THF was added with vortex-mixing ([DPHl/[lipidl = V1000). Samples were incubated above the phase transition temperatures (45"C for DPPC, 30 "C for DMPC) for at least 1h. Fluorescence spectroscopy was recorded on a JACSO FP-770spectrofluorometerequippedwith polarizers and connected with a thermocontrolledwater bath. DPH was excited at 357 nm and fluorescence was detected at 430 nm. The temperature inside the cuvette was monitored with a thermocouple digital thermal meter. Heating and cooling rates were approximately 10 " C h All the samples were measured for at least one cooling run and one heating run with the cooling run from a temperature above the phase transition temperature, and the data here were the average of two or three measurements.12 Differential Scanning Calorimetry (DSC). Multilamellar vesicles of different lipid (or mixtures) samples for DSC measurements were prepared by thorough hydration of the CH3OH-cast films of DPPC with the required molar ratios of 1,2,or 3 in deionized water at 50-55 "C for 10 min, followed by vortexmixing the samplesfor 5 min and exposureto two cycles of freezethawing with dry ice-methanol as the coolant. The final total concentrationof the suspensionswas 40 mM. The DSC analysis was performed on a Seiko 120 DSC from 10 to 60 "C with a scan rate of 0.8 "C/min.
Results and Discussion Characterization of the Aqueous Suspensions of Lipid Mixtures. When 1, 2, or 3 were sonicated with DPPC or DMPC in aqueous media, transparent suspensions were obtained. The average hydrodynamic diameters were 48 f 14 nm (Coulter, N4SD), which could be regarded as small unilamellar vesicles (SUV).An increase in the ratio of 1,2, or 3 u p to 50 mol % did not change the size of the mixed vesicles obviously. Owing to their high compatibility with lipids, they can all be homogeneously incorporated in the phospholipid assembly like cholesterol and the vesicles containing 1,2, or 3 showed high stability toward aggregation and fusion. (12) Iwamoto, K.; Sunamoto, J.; Inoue, K.; Endo, T.; Nojima, S. Biochim. Biophys. Acta 1982, 691, 44.
OSS
3
r-----l r7
A
I
5 (2)
0 0.2
0
a
I
0
20
I
I
40
I
I
I
60
Tlmr (hr)
Figure 1. Time-dependentturbidity changes of DPPC vesicles containing 50 mol % cholesterol or various amount of 2 at 4 "C. For example, 2 was used to evaluate the stability of the
mixed vesicles toward aggregation by a turbidity measurement at 4 "C. As shown in Figure l, DPPC SUV prepared by sonication aggregated with a rapid increase in A O.D. This is a well-known phenomenon caused by the constrained lipid packing in the bilayer membrane with high curvature of the bilayer membrane.13 The vesicles containing 50 mol % cholesterol showed higher stability toward aggregation because incorporation of cholesterol can improve the lipid packing by decreasing the curvature. For the vesicles mixed with 2, high stability was also observed even at an incorporation ratio of 5 mol %.
There may be two reasons for such a significant effect of 2 on the dispersion state of the mixed vesicles. One is that incorporation of 2 can make the surface potential positive, and the electrostatic repulsive force inhibits the aggregation of the dispersion; the other reasonable explanation may be that the enhancement of the molecular motion in the mixed vesicles (see below) increases the disaggregation rate of the vesicles (equivalent to the effect of temperature elevation). Destabilization of vesicular suspension by incorporation of the lithocholic acid derivatives was not observed. The utilization of membranespanning lipids may be a good method to obtain a stable lipid bilayer membrane; there have also been some other reports about the enhancement of the thermal stability of vesicles by membrane-spanning lipids.8-11 'H-NMR Measurements. The temperature-dependent lH-NMR spectra of DPPC vesicles are shown in Figure 2a. Obvious differences can be observed between the spectra, especially those below and above the phase transition temperature (T,= 40 "C). It is well-known that the lH-NMR spectrum of vesicle suspensions is qualitatively different from that of a homogeneous organic solution of the corresponding lipid because of the strong interaction among m01ecules.l~The relative peak intensity ratio of hydrocarbon groups to that of choline head groups can be used to measure the phase transition temperature, usually with an abrupt change near T,. Resonance peaks attributed to the hydrocarbon chains (1.2ppm) disappeared below the T,, indicating that the mobility of hydrocarbon chains was seriously restrained in the gel state. On the other hand, peaks ofcholine methyl groups (3.2ppm), which located on the surface of the vesicles, were not directly influenced by the packing state of hydrocarbon chains; the peaks (3.2ppm) still appeared even below T,. Figure 2b shows the lH-NMR spectra of DPPC/ cholesterol (50/50, mol/mol) vesicles. For the choline groups, there was no significant difference either below (13)Wong, M.; Anthony, F. H.; Tillack, T. W.; Thompson, T. E. Biochemistry 1982,21, 4126. (14)Wennerstrom, H.; Linddblom, G. Q.Rev. Biophys. 1977,10,67.
Li et al.
3164 Langmuir, Vol. 11, No. 8, 1995
--
0.3
60.2
-
8
-
e
I
qo.1
-
-
mol% In DMPC
mol% In DPPC 15 (3)
-25
(3)
-
0.0
'
'
1
1
'
1
'
!
'
1
'
1
'
1
'
n
--'-A&/
1
4
a
z
b (PPm)
1
0
,
1
a
z
I
1
0
8 (PPW
Figure 2. Temperature-dependent lH NMR spectra of DPPC (a),DPPC/cholesterol(50/50,mol/mol) (b), DPPC/2 (85/15,mol/ mol) (c), and DPPC/2 (50/50, mol/mol) (d) vesicles.
or above T, compared with DPPC vesicles, because cholesterol had little effect on the head group of phosph01ipid.l~ However, above T,,relatively small lH-NMR peak intensities compared with those of DPPC vesicles at the same temperatures indicates that the mobility of the hydrocarbon chains was restricted significantly by the four rigid flat rings of cholesterol. It is suggested that incorporation of cholesterol could improve the packing of lipid molecules, and thus the mobility of lipid molecules was reduced. Another important phenomenon is t h a t the peak intensity of the terminal methyl groups (0.8 ppm) can be even higher than that of the methylene groups (1.3 ppm) a t temperatures above the T, of the host DPPC membrane, though the relative number of the methylene groups is much more than that of the methyl groups in the DPPC/cholesterol mixed system. Although this may be caused by the tail chain of cholesterol with high flexibility, we favor the explanation that in DPPC/ cholesterol vesicles, cholesterol molecules locate their rigid steroid rings near the surface and restrict the mobility of the methylene groups of DPPC hydrocarbon chains. They do not affect the mobility of acyl chains near the center of lipid bilayer; therefore, the mobility of the terminal methyl groups of DPPC becomes higher than that of the methylene groups with increasing temperature. On the other hand, interesting results were observed for the DPPC vesicles containing 15 and 50 mol % of 2 as shown in parts c and d of Figure 2, respectively. The same results were obtained for 1 and 3. It can be seen that the NMR peak shape of the methylene and methyl groups of DPPC/2 vesicles is different from those of DPPC and DPPC/cholesterol vesicles; almost the same relative peak intensity of methylene and methyl groups was observed. Below the T,of the host DPPC membrane, the relatively high IH-NMR peak intensity of the methylene groups revealed the higher mobility of lipid molecules in the DPPC/2 vesicles than in the DPPC/cholesterol vesicles. On comparison ofparts c and d of Figure 2, it is not difficult ( 1 5 ) Brown, M. F.; Seelig, J. Biochemistry 1978,17, 381.
(16)Oldfield, E.;Meadows, M.; Rice, D.; Jacobs, R. Biochemistry 1978, 17, 2727.
20
30
40
50
Temperature ( ' C )
5
25 35 Temperature (OC)
15
5
Figure 3. Plots of fluorescenceanisotropy ( r )vs temperature for DPPC vesicles of various 3 molar concentrations (a)and 50 mol % of 1, 2, and 3 (b). (c and d) DMPC vesicles. to see that the mobility of hydrocarbon chains increased with the ratio of 2 in the mixed vesicles. When 50 mol % of 2 was incorporated into the DPPC bilayer membrane, the mobility of the hydrocarbon chains was so high that the lH-NMR peaks of methylene (1.2 ppm) and the terminal methyl groups (0.9 ppm) were obvious even a t room temperature, far below the T,(43 "C) of the DPPC membrane. In Figure 2c, the abrupt change in peak intensities of the choline methyl group (3.2 ppm) from 30 to 35 "Ccorresponds to the phase transition of the DPPC vesicles containing 15 mol % of 2; however, in Figure 2d, no such clear phase transition was observed. The three lithocholic acid derivatives were designed to bridge the lipid bilayer membrane and locate their bulky steroidal moieties just in the center of DPPC bilayer membrane. The other packing state besides the membrane-spanning structure in the bilayer membrane would be the backfolded structure. However, this structure is generally observed for the molecules having no rigid group a t the center of the molecule. Therefore, the bulky steroidal group should disturb the packing of the lipid molecules in the center of the lipid bilayer, resulting in higher mobility of the terminal methyl groups below T,. This effect was quite different from t h a t of the steroidal moiety in the DPPC/cholesterol vesicles due to the difference in location in the bilayer. Fluorescence Depolarization Method. DPH is a probe frequently used to monitor the fluidity around the hydrophobic portion of the bilayer membrane and to detect the gel-liquid crystalline phase transition temperature in these systems.I7 When DPH was used for the fluorescence depolarization study of the cholesterol effect on the molecular mobility in the bilayer membrane, it was confirmed that cholesterol disordered the hydrocarbon chain packing below T, and decreased the molecular motion above T,. No clear phase transition behavior existed when the concentration of cholesterol in lipid membrane was 50 mol %.la The mixed vesicles of 3 and DPPC showed a similar tendency to those of DPPC and cholesterol vesicles, but the degree of the effects differed as shown in Figure 3a. Below the main transition temperature of the host DPPC bilayer membrane, the anisotropy ( r ) of DPPC/3 mixed vesicles decreased significantly with increasing the ratio (17)Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978,515, 367. (18)Li, Z.-C.; Li, F.-M.; Arase, S.; Takeoka, S.; Tsuchida, E. Chem. Lett. 1994,2199.
Langmuir, Vol. 11, No. 8, 1995 3165
Lithocholic Acid Derivatives of 3 in DPPC vesicles, while only a slight increase in r could be observed above T,, in comparison with those of DPPC/cholesterol mixed vesicles. Clear phase transition behavior also disappeared a t 50 mol % of 3. These results can also be interpreted as the incorporation of the bulky steroidal moiety into the bilayer membrane below the T, obviously decreases the orientational order of the phospholipid hydrocarbon chains, and thus DPH can move more freely. The mobility of the hydrocarbon chains might be slightly restricted by incorporation of 3 in DPPC membrane above the T,in comparison with cholesterol. Different structure of the steroidal moieties between lithocholicacid derivatives (coprostan type)and cholesterol (cholestane type) might be the cause of this phenomenon. When the effect of these three molecules with different chain lengths is compared at the same concentration of 50 mol %, similar results with some differences can also be seen as shown in Figure 3b. Below the T,of the host DPPC membrane, the disruptive efficiency of these three molecules was in the order of 3 > 2 > 1;above the T,,they all slightly increased the packing of the lipid in bilayer membrane, but 3was less effective. Although it is difficult to explain the exact reason for this phenomenon, it might be caused by matching or mismatching of the hydrocarbon chain length to the thickness of the gel-state DPPC bilayer. 2 was designed to have almost the same length as the thickness of the membrane, while 1 and 3 are longer and shorter than the thickness, respectively. The incorporation of 3 into the membrane would be unfavorable because it is shorter than the bilayer thickness. Chain segments of DPPC molecules near the center of the bilayer would exist in more gauche conformations in order to shorten the bilayer thickness. As a result, the mobility of the hydrocarbon chains increases, in addition to the effect of the steroidal moiety. This will be the reason why the incorporation of 3 into the DPPC membrane was much more effective than the other two (1 and 2). In the case of 1,because the total length of the molecule is longer than the thickness of DPPC bilayer membrane, the packing state ofthe steroidal moiety in the membrane should change significantly in order to adjust the total molecular length to the bilayer thickness, with the hydrophobic chain orienting parallel to the DPPC acyl chains. Therefore, the effect ofthe bulky steroidal moiety of 1 may not be as strong as that of the other two mixed vesicles, resultingin its relative small effect on the mobility of alkyl chains in the membrane. In order to study the effect of these litholic acid derivatives on the membranes in more detail, DMPC, which has a bilayer thickness and phase transition temperature (23 "C) different from DPPC, was also used as the host phospholipid in bilayer membrane. Results were similar to those with DPPC vesicles as shown in parts c and d of Figure 3. Mainly, the phase transition temperature shifted to the lower side with the ratio of 3. Because 3 is almost the same length as the DMPC bilayer thickness, and 2 is longer, the effect of 2 on the phase transition is less than that of 3. Phase Transition Study by DSC. In order to monitor the thermally induced phase transition of the mixed vesicles, DSC was applied to DPPCA mixed vesicles, and the results are shown in Figure 4a. The endothermal peak of the main phase transition of the pure DPPC membrane (40.6 "C) was broadened and moved to a lower temperature (37.8 "C) even with a 5 mol % incorporation of 2. If the ratio of 2 was gradually increased, the peak of the main phase transition appeared a t a lower temperature over a broader temperature range, and no obvious phase transition was detected when 2 reached 50 mol %. From the results obtained by NMR and fluorescence
20
30
40
50
Tamprratura ('C)
Figure 4. DSC thermograms for DPPC vesicles of various 2 molar concentrations (a) and 15 mol % of 1, 2, and 3 (b). depolarization studies, it was clear that this was due to the effective membrane fluidizing effect of 2 on the gel state of the DPPC membrane. Furthermore, this behavior is similar to that in a DPPC/ cholesterol mixed systems, but some points are quite different. The effect of cholesterol on the thermotropic phase behavior has also been studied systematically and extensively using DSC.lg Although the results reported by different laboratories do not coincide well with each other, some main results are considered to be the same. They include (1)broadening of the cooperative gel-to-liquid crystalline phase transition of the phospholipid bilayer with a slight shiR to the higher side as the concentration of cholesterol was increased and (2) the phase transition completely disappearing when the ratio of cholesterol was 50 mol %. Our lithocholic acid derivatives significantly shifted the transition temperature to the lower side. It is a well-known phenomenon that alternation in the lipid head group results in altered packing density of the head groups and consequently a change in the phase transition behavior of the membranes;20 therefore, one may argue that different head groups of 2 from DPPC may be one reason for such a remarkable influence of 2 on the phase transition behavior of the host phospholipid membrane. However, this cannot be the case according to recent reports on the interactions of phosphatidylcholine with cationic quaternary ammonium amphiphiles. A head group difference affects the packing state of the hydrophilic region with little effect on the hydrophobic region.21 Therefore, a n increase in the mobility of the hydrophobic chains in the DPPC/2 vesicles should be caused by the interactions of 2 and DPPC in the hydrophobic region. Because the phase transition occurs from the center of the bilayer membrane to the region near the surface, only a small amount of 2 would be sufficiently effective enough to influence the lipid packing in the center of the bilayer due to the bulky steroidal moiety, resultingin a significant change in the phase transition temperature. It should be pointed out that even when 50% of 2 was incorporated into DPPC membrane, no obvious phase separation was (19) McMullen, T. P. W.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1993,32,516. (20) Edwards, K.;Almgren, M. Langmuir 1992,8,824. (21)Scherer, P.G.;Seeling, J. Biochemistry 1989,28,7720.
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observed as in the case of the other mixed vesicles.22This should be attributed to the good compatibility of 2 with DPPC . To study the effect of hydrophobic chain length on the phase transition, the three molecules were incorporated a t the same ratio (15 mol %) into DPPC vesicles and the multilamellar vesicles were studied by DSC as shown in Figure 4b. At this ratio, they can all significantly influence the phase transition behavior of the host DPPC membrane; especially the effects of 2 and 3 were much greater than that of 1 with lower and broader phase transition peaks. Although the main peak ofthe DPPC/3 vesicles was almost the same as that of the DPPC/2 vesicles, the peak intensity which reflected the AH ofthe phase transition was smaller. This indicates that 3 was much more effective than 2 for disruption of the gel state lipid packing of the host DPPC membrane. These results are in good agreement with the above mentioned results obtained by the fluorescence depolarization method, which were also interpreted based on a different packing state in the lipid bilayer membrane. (22) Chapmann, N. F.; Owens, N. F.; Phillips, M. C.; Walker, D. A. Biochim. Biophys. Acta 1969, 183, 458.
Conclusions Incorporation of a steroidal moiety in the center of a lipid bilayer membrane using membrane-spanning lithocholic acid derivatives obviously increases the mobility of the lipid bilayer membrane and shifts T,to the lower temperature side in comparison with cholesterol. The lithocholic acid derivatives which are shorter than the DPPC bilayer thickness disturb the lipid packing more intensively. The resulting mixed vesicles showed a higher stability of the dispersion state. This remarkable phenomenon stimulated our further research in this specific field.
Acknowledgment. This work was done under a cooperative research project between Waseda University and Peking University and was partially supported by a Grant-in-Aid for Scientific Research (No.05650930) from the Ministry of Education, Science and Culture, Japan. Z.-C. Li thanks Peking University for a n academic leave. LA9409135
