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Langmuir 1995,11, 912-916
Molecular Interactions between Lipids and Some Steroids in a Monolayer and a Bilayer. 2 Yoshiko Takao,t Hitoshi Yamauchi,+Jiradej Manosroi,$Aranya Manosroi,* and Masahiko Abe*ptp§ Faculty of Science and Technology, Science University of Tokyo, 2641, Yamazaki, Noda, Chiba 278, Japan, Department of Pharmaceutical Technology, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand, and Institute of Colloid and Interface Science, Science University of Tokyo, 1-3, Kagurazaka, Shinjuku, Tokyo 162, Japan Received June 6, 1994. I n Final Form: November 10, 1994@ Molecular interactions between L-a-dipalmitoylphosphatidylcholine(DPPC) and some steroids having different hydrophilicgroups in a monolayer and in a bilayer are investigated in terms of surface pressure, permeability, and microviscosity. The steroids used in this study are stigmasterol, 4,22-stigmastadien%one (stadiene), and stigmasterol acetate (acetate). The collapse pressures for the mixed monolayer of DPPC with various steroids depend upon the molar ratio ( X )and types of hydrophilic groups in steroids. A one-stage collapse is observed for X 5 0.5 of stigmasterol, X I0.3 of stadiene and acetate, whereas a two-stage collapseis seen in other molar ratios of the steroids. The mole fraction within which liposomes are formed is dependent upon the types of hydrophilicgroup within the steroids. In comparing monolayer and bilayer membranes (liposomes),liposomes are formed at the mole fraction of steroids which have a one-stage collapse in DPPC/steroid mixed monolayer. The permeability of the bilayer membrane decreases with increasing cohesive force between DPPC and steroids, but increases with a decrease in microviscosity near the hydrophilic portions, in the following order: stigmasterol < stadiene < acetate. This study demonstrates that the permeability of liposomes is dependent upon the hydrophilic group of the steroids, and the stability of the liposomes increases with an increase in the cohesive forces between DPPC and the steroids.
Introduction It is now recognized that liposomes, consisting of lipid bilayer membranes, are of great importance as a valuable experimental tool for examination of biological membranes and as carriers for drug delivery systems (DDS).1-3 Normally, liposomes are composed of a phospholipid, dicetyl phosphate (a charged lipid), and cholesterol (a steroid which serves to prevent coagulation)in liposomes to minimize leakage of the entrapped material^.^-^ Many investigations have been performed on the pharmacological aspects of liposomes in vivo and/or in vitro. Only few studies have been done, however, on the physicochemical properties of liposomes, particularly, the molecular interactions between phospholipids and steroid molecules in a bilayer membrane. Investigation of these interactions is of great importance not only for practical use, but for theoretical purposes as well. There are many other steroids besides cholesterolwhich can be incorporated into liposomes; however, the chemical structural effects of these steroids on the stability of liposomes has not yet been clarified, although it is k n o w n that molecular interactions, in particular two-dimensional interactions, between phospholipids and steroids in liposomes play a very important role in the stability of their membranes. Faculty of Science and Technology,Science University of Tokyo. Department of Pharmaceutical Technology, Chiang Mai University. Institute of Colloid and Interface Science, Science University of Tokyo. Abstract published in Advance ACS Abstracts, February 1, 1995. (1)Bachhawat, B. K. InLiposome Technology; Gregoriadis, G., Das, P. K., Ghosh, P., Eds.; CRC Press, Inc.: Boca Raton, FL, 1984; Vol. 3,
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p 118. (2) Hirano, J.; Hibino, E. J . Jpn. Oil Chem. SOC.1988,37,864. (3) Kitano, H. J . Surface 1987,25,231. (4) Sunamoto, J.;Shironita, M. J . Surface 1979,17, 357. (5) Yamauchi, H.; Kikuchi, H. Fragrance J . 1987,87, 68. (6) Kikuchi, H.; Inoue, K. Saibokogaku 1983,12,61. (7) Kikuchi, H.; Inoue, K. J . J p n . Oil Chem. SOC.1985,34,785. (8) Akutsu, H.; Kyogoku, Y. J . Seikagaku 1982,54, 1048.
In a previous paper,g the chemical structural effects of steroids on the molecular interactions of hydrophilic groups in DPPChteroid mixed systems was investigated. It was demonstrated that the molecular interaction between DPPC and steroids increases with a decrease in the number of side chains and double bonds of the steroid molecules. According to the measurements of permeability, polarity, and fluidity of the bilayer membranes, the stability of the bilayer membrane has been found to depend upon the hydrophobic interactions. In this paper, we report the chemical-structural effects of hydrophilic groups in steroids on the surface pressure of a monolayer membrane and on permeability as well as the microviscosity of a bilayer membrane. Molecular interactions between DPPC and the three types of steroids with different hydrophilic groups in a monolayer and in a bilayer membrane will be discussed.
Experimental Section Materials. Phospholipid. L-a-Dipalmitoylphosphatidylcholine (DPPC,99.6%)was supplied from Nippon Oil and Fats Co., Ltd., Tokyo, Japan, and used without further purification. Steroids. Stigmasterol and 4,22-stigmastadien-3-one (stadiene) were purchased from Sigma Chemical Co., St. Louis, MO. The steroids were recrystallized from ethanol and analyzed by differential scanning calorimetry. Stigmasterolacetate (acetate; 99.0%pure) and cholesterol (99.9% pure) were purchased from Sigma Chemical Co. and used without further purification. ChargedMaterials. Dicetylphosphate (DCP,99.6%pure) was
purchased from Sigma Chemical Co. and used without further
purification.
Fluorescence Probes. 2-(9-Anthroyloxy)palmiticacid (2AP;98% pure)was purchased from Sigma Chemical Co. and used without further purification. Water used in this experiment was twice distilled and deionized using an ion-exchange instrument (NAN0 pure D-1791 manufactured by Barnstead Co., Ltd., Boston, MA). Its resistivity was approximately 18.0 M Q and its pH was 6.7. Chloroform (9) Yamauchi, H.; Takao, Y.; Abe, M.; Ogino, K. Langmuir 1993, 300, 9.
0743-7463/95/2411-0912$09.00/00 1995 American Chemical Society
Interactions between Lipids and Steroids (99.0%) from Wako Chemical Industries, Ltd., was used after distillation. Glucose was purchased from Nippon Rikagakuyakuhin Co., Ltd., Tokyo, Japan. Phosphate-buffered saline (PBS) was purchased from Nissui Pharmaceutical Co., Ltd., Tokyo, Japan. All were commercial products of reagent grade. Method. Measurement o f Surface Pressure. Surface pressures (F dyne/cm)of DPPC/steroid mixed monolayers at the airwater interface were determined by a Wilhelmy plate method using a surface pressure meter of type HBM-A (Kyowa Interface Science Co., Ltd., Tokyo, Japan) with a bar made from Teflon. The mixtures of DPPC and steroids in various molar ratios were dissolved in chloroform; the total lipid concentration was 1.5 x m o m . After the lipid-chloroform solution was spread on the water without causing surface disturbance using a microsyringe, the system was allowed to stand for 15 min. The speed of compression on the monolayer was 20 m d m i n , considering the absorption equilibrium of lipids at the surface. The experiments were carried out at 35 "C. The pH was 6.7. Preparation of Liposomes. Liposomes, which were reversephase evaporation vesicles (REV), were prepared by a conventional technique similar to the method of Szoka and Papahadjopoulos.l0 Briefly, DPPC and DCP were dissolved in a mixture of diethyl ether and ethanol and then a 0.28 m o m glucose solution was added in a proper volume. A water in oil (W/O) emulsion was formed by sonication (bath type; Branson B-220, Tokyo, Japan) for 10-20 min. After the solvent of the emulsion was removed using a rotary evaporator, a gel formed. Finally, REV were obtained after being shaken on a Vortex mixer several timesg Liposomes were extruded at 55 "C through a polycarbonate membrane filter of 0.2 pm pore size according to the method of Olsen et a1.l1 Quantification of DPPC and Cholesterol. The concentrations of DPPC and cholesterol were determined by enzymatic assay12 using a phospholipids-B-test Wako and a cholesterol-C-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Measurement of Permeability of the Bilayer Membranes. In order to estimate the permeability of liposomal membranes prepared with various steroids, the time course of glucose leakage from the liposomes was determined. Briefly, glucose, which was the aqueous model material, was encapsulated into liposomes, and the unencapsulated glucose was separated from the liposomes using a dialysis technique with a cellophane tube (Union Carbide Co., Chicago, IL). The liposomes were dialyzed in a cellophane tube with 1 L of saline for 3 h at about 5 "C; the saline was changed three times during dialysis. This method and conditions are commonly used to separate the unencapsulated materials. Then, the amount of glucose encapsulated in the liposomes was determined. Glucose was first extracted into a water phase from liposomes, according to the procedure of Blight-Dyer,13and then assayed by the phenol-sulfuric acid method14 using a spectrophotometer (type MPS-2000, Shimadzu Co., Ltd., Kyoto, Japan). Measurement of Microviscosity of the Bilayer Membranes.
DPPC, steroids, DCP, and the appropriate amount of 2AP were dissolved in chloroform in a test tube. The solvent was then removed by flowing nitrogen gas into the test tube. The residual solvent was further dried in a desiccator under vacuum overnight a t room temperature. Four milliliters of PBS was added to the lipid film and then warmed (55-60 "C) above the phase transition temperature of DPPC ( T , = 41 "C) for 30 min. The test tube was shaken vigorously on a Vortex mixer and a more homogeneous liposome dispersion was obtained using a 10 min sonication. The total lipid concentration was 10 mM and the molar ratio of 2AP to the total lipids is 1:300. The microviscosity of liposomes was determined according to the intensity of 2AP using a Fluorescence spectrophotometer type (RF-540; Shimadzu Co., Ltd., Kyoto, Japan) at the excitation and emission wavelengths of 360 and 440 nm, respectively. Here, the fluorescence polarization (P)is defined as (10)Szoka,F., Jr.;Papahadjopoulos,D. Proc. Natl.Acad. Sei. U S A . 1978.75. - , 4194. (11)Olsen, F.; Hunt, C. A.; Szoka,F., Jr.; Vail,W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1978,557,9. (12)Takayama, M.; Itoh, S.; Nagasaki, T.; Tanimizy, I. Clin. Chim. Acta 1977,79,93. (13) Blight, E. G.; Dyer, W. J.; Can. J . Biochim.Phys. 1969,37,911. (14)Dubois, M.;Gilles, K. A.; Hamilfon, J. K.; Rebers, P. A.; Smith, F. Anal. Chem. 1956,28,550. ~~
- >
~~~
~
Langmuir, Vol. 11, No. 3, 1995 913 50 1
"0
10
DPPC:sterol
30 40 50 60 70 80 Area per molecule (A %nolecule)
20
90
100
Figure 1. The surface pressure (n)-area (A) curves of DPPC/ stigmasterol mixed monolayers at 35 "C.
where ZII is the fluorescence intensity polarized parallel to the direction of polarization of the excitation beam, Zi is that perpendicular to it, and G is the factor t o correct for instrument anisotropy.l5 All experiments were carried out at 35 "C.
Results and Discussion Behavior of Mixed Monolayers of Lipids at the Air-Water Interface. We have previously reported that several kinds of steroids having different side chains such as cholesterol,P-cholestanol,p-sitosterol, stigmasterol, and 4,22-stigmastadien-3-one can affect lateral interactions between DPPC and steroids in a mixed monolayer.16 Figure 1 shows the plot of surface pressure (n)vs molecular area (A) of DPPC/stigmasterol mixed monolayers in various molar ratios demonstrating that each DPPC/stigmasterol mixed monolayer forms a condensed monolayer at various molar ratios. The n-A curve of the monolayer of DPPC alone shows a transition from a n expanded state to a condensed state a t 30 mN/m. With a n increasing molar ratio of stigmasterol in the monolayer, this transition cannot be observed on the n-A curve. A collapse stage of a mixed monolayer is found to depend on the molar ratios of steroid to DPPC. When compressed, the mixed monolayers of DPPC/stigmasterol at 10:0,7:3, 5 5 , and 0:lO show a one-stage collapse, while the mixed monolayer of DPPC/stigmasterol a t 4:6 and 3:7 show a two-stage collapse. The collapse of the monolayer seems to be a phase transition from a two-dimensional phase to a threedimensional one. Nakagaki et al.17 and Demel et al.Is have suggested that a two-stage collapse reflects the phase separation in a mixed monolayer. It is also well-known that, when a collapse pressure and/or equilibrium spreading pressure of the monolayer are dependent upon molar ratio of mixtures, one can discuss the miscibility of two ingredients in a monolayer phase (two-dimensionalphase) and/or collapsed phase (three-dimensional phase).19,20 Figure 2 depicts the collapse pressure-mole ratio ( X ) curves of the DPPC/stigmasterol mixed monolayer; Figure (15) Chen, R. F.; Bowman, R. L. Science 1965,147,792. (16)Ogino, K.;Goto, M.; Abe, M. J . Jpn. Oil. Chem. SOC.1988,37, 647. (17)Nakagaki, M.;Handa, T. Bull. Chem. SOC.Jpn. 1976,49,880. (18)Demel, R. A,; Bruckdorfer,K. R.; Van Deenen, L. L. M.; Biochim. Biophy. Acta 1972,255,311. (19)Zsako, J.; Tomoaia-Colisel,M.; Chifu, E.; J . Colloid Interface Sci. 1984,102,186. (20) Nakagaki, M.; Funasaki, N.; Bull. Chem. SOC.Jpn. 1974,47, 2094.
914 Langmuir, Vol. 11, No. 3, 1995
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0
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0.2 0.4
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Takao et al.
1.0
Area per molecule (A '/molecule)
Figure 2. The collapse pressure-mole fraction ( X ) curves of DPPC/stigmasterol mixed monolayers at 35 "C.
2 was obtained from Figure 1. M here denotes a monolayer phase (two-dimensionalphase) and S denotes a collapsed phase (three-dimensionalcollapsed phase). The collapse pressure of monolayer from the DPPC alone is larger than that of stigmasterol alone. The collapse pressures (0)of the DPPChtigmasterol mixed monolayer obtained from the n-A curves are slightly larger than its equilibrium spreading pressure (0). There is a progression from a one-stage collapse to a two-stage collapse when the molar ratio of stigmasterol increases up to 0.5. Moreover, in the region (Xstlgmasterol 5 0.5) of the one-stage collapse, the collapse pressure is almost independent of the increase in molar ratio of stigmasterol. This means that stigmasterol is completely immiscible with the monolayer phase. On the other hand, in the region of the two-stage collapse (Le., Xstlgmasterol > 0.51, the lower collapse pressure is dependent upon the increase in stigmasterol molar fraction. This indicates that DPPC molecules are miscible with stigmasterol in the monolayer phase. Nakagaki et a1.21 and J O O have S ~ ~suggested that an equilibrium spreading pressure indicates the miscibility in a collapsed phase. For a DPPChtigmasterol system, the equilibrium spreading pressure does not depend upon a n increase in the molar ratio of stigmasterol. Therefore, it appears that the DPPC molecule is immiscible with stigmasterol in the collapsed phase of all molar ratios. For a two-stage collapse, when a mixed monolayer is compressed, one of the components (stigmasterol) starts separating a t lower collapse pressure, which is the first collapse point. This separation continues during the further compression, accompanying the increase in collapse pressure and the approach of the value of the mole fraction in the insoluble component to that of the concollapse monolayer. As the collapse pressure reaches higher collapse point, the other component (DPPC) starts separating simultaneously, but this second deposit does not mix with the first one. In order to investigate the effect of hydrophilic groups in steroids on the mixed monolayers, various steroids (i.e., stigmasterol, stigmasterol acetate, and 4,22-stigmastadien-3-one) are used in this experiment. Figures 3 and 4 are n-A curves and the collapse pressure-X curves of the DPPCIacetate mixed monolayers. Figures 3 and 4 demonstrate that in spite of molar ratios, the mixed monolayers form a condensed monolayer. When the mixed monolayer is compressed, a one-stage collapse is observed a t the molar ratios of DPPC/acetate a t 10:0, 7:3, and 0:10, while a two-stage collapse is seen a t 6:4, 5:5, and 3:7. (21)Nakagaki, M.; Funasaki, N.; Bull. Chem. SOC.Jpn. 1974, 47, 2482. (22) Joos, P. J . Colloid Interface. Sci. 1971,35, 215.
Figure 3. The surface pressure (n)-area (A) curves of DPPC/ 4,22-stigmastadien-3-one mixed monolayers at 35 "C.
6.0 0.2 0.4
0.6
0.8
1.0
Xstadiene
Figure 4. The collapse pressure-mole fraction (X)curves of DPPC/4,22-stigmastadien-3-one mixed monolayers at 35 "C. DPPC:acetate
Area per molecule (A '/molecule)
Figure 5. The surface pressure (n)-area (A) curves of DPPC/ stigmasterol acetate mixed monolayers at 35 "C.
For a DPPCIstadiene mixed monolayer, as shown in Figures 5 and 6, a similar tendency is indicated. However, the compressibility of a DPPUacetate mixed monolayer is larger than that ofDPPC/stigmasterol a t the same molar ratio. We found that compressibility is dependent on the types of steroids in the following order: stigmasterol < acetate < stadiene. The explanation for this is that the orientation and packing of steroids in the mixed monolayer a t the air-water interface is dependent upon the hydrophilic groups of the steroids, as previously m e n t i ~ n e d . ~ As is evident from Figures 4 and 6, each collapse pressure of the monolayer of steroid alone is smaller than that of stigmasterol. Therefore, the difference between the collapse pressure of DPPC and steroids is found to be dependent upon the hydrophilic groups of the steroids in the following order: stigmasterol < stadiene < acetate.
Interactions between Lipids and Steroids
Langmuir, Vol. 11, No. 3, 1995 915
-0
k
P -; 0
30 20 10
0.2 0.4
0.0
0.6
0.8 1.0
Xaeetate
Figure 6. The collapse pressure-mole fraction (X)curves of DPPC/stigmasterol acetate mixed monolayers at 35 "C.
Area per molecule (A '/molecule)
Figure 8. The surface pressure (n)-area (A) curves of DPPCI cholesterol mixed monolayers at 35 "C.
. A
E 2
E
701 0.0
I
0.1
I
0.2
I
0.3
1
0.4
I
0.5
I
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'stemid
Figure 7. The relationship between the formation of liposome and mole fraction of steroids: 0 ,stigmasterol;(3, stigmasterol acetate; 0, 4,22-stigmastadien-3-one.
The collapse pressure is larger than the equilibrium spreading pressure as is the case with DPPC/acetate and/ or DPPWstadiene mixed monolayers. Therefore, the difference between the collapse pressure (0) and equilibrium spreadingpressure (0)is consistent with the order of collapse pressures. It is suggested that the cohesive force between DPPC and steroids increases with a decrease in the difference between collapse pressure and the equilibrium spreading pressure, because the collapse pressure represents a supercompressive state. The cohesive force then would be dependent upon steroids in the following order: acetate < stadiene < stigmasterol. In DPPUstadiene and DPPC/acetate mixed monolayer systems, the collapse changes from one-stage (0= one stage) to two-stage (0 = two stage) upon increasing the molar ratio of the steroids. The transition is found to be more than 0.3. The behavior of these steroids in monolayers is quite similar to that of stigmasterol. We found that, in mixed monolayer systems comprised of DPPC together with various steroids, the property of DPPC is maintained a t a one-stage collapse, but not at a two-stage collapse. In addition, the cohesive force between DPPC and the steroids is dependent upon the hydrophilic groups of the steroid in the following order: acetate < stadiene < stigmasterol. Correlationbetween a Monolayer and a Bilayer. The relationship between the formation of liposomes and the mole fraction of steroid vs mole fraction of steroid in the liposomes is shown in Figure 7. It shows the plot of ratios of DPPC concentration in liposomes against the addition of DPPC concentrations. An amount of 100% DPPC is used for liposome formulation with molar ratio of steroids of less than 0.5 for stigmasterol and less than 0.3 for stadiene and acetate. With larger molar ratios than these, small amounts of DPPC and steroids separate out and precipitate in the system.
40 30 20 10 50
0 0.0
1
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1
1
0.2
0.4
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Xchol
Figure 9. The surface pressure (n)-mole fraction (X,curves of DPPC/cholesterol mixed monolayers at 35 "C: 0, collapse
pressure; 0 , equilibrium spreading pressure.
As is evident from Figures 2,4,6, and 7, a relationship exists between liposome formation and collapse in the mixed monolayer. Liposomes are not formed at all when the two-stage collapse occurs in a monolayer. These results seem to center around the DPPC properties a t one-stage collapse in the monolayers. Moreover, these results, which show that DPPC is not miscible with steroids a t one-stage collapse in the monolayer, agree with those of cholesterol, which forms in clusters in liposomal membranes.23 Figures 8 and 9 depict n-A curves and the collapse pressure-X curves of DPPC/cholesterol mixed monolayers. The behavior of cholesterol in the mixed monolayers is similar to that of stigmasterol. The transition molar ratio between the one-stage and two-stage collapse is 0.5. Figure 10 shows the relationship between the percentage of DPPC and cholesterol in liposomes and the molar ratio of cholesterol. The precipitation of DPPC and cholesterol is increased with increasing molar ratios of cholesterol from 0.5. The two-stage collapse is observed a t more than 0.5. Permeability of Bilayer Membranes. In order to investigate the effect of hydrophilic groups in steroids on the stability of bilayer membranes, the permeability of bilayer membranes was measured. Figure 11represents the time course of glucose leakage from bilayer membranes prepared with a 0.3 mole fraction of steroid. In fact, it is possible to prepare liposomes with other mole fractions of these steroids. Cholesterol is uniformly distributed in the bilayer membrane a t molar (23) Yotsuyanagi, T.;Hashimoto, H.; Iwata, M.; Ikeda, K. Chem. Pharm. Bull. 1987,35, 1228.
Takao et al.
916 Langmuir, Vol. 11,No. 3, 1995 75 DPPC:steroid:DCP = 7:3:1
7o Temp.35.C
41
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s - 05
45 50 0.0
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Xehol
Figure 10. The relationship between liposome formation and mole fraction of cholesterol: 0, DPPC; 0 , cholesterol.
Incubation time (hours) Figure 11. Time dependence of glucose leakage from a bilayer membrane at 35 “C. (DPPC/steroid/DCP= 7:3:1):0,without steroid; 0 , stigmasterol acetate; 0, 4,22-stigmastadien-3-one; 0 , stigmasterol; 8 , cholesterol. The data of cholesterol are replotted from ref 9.
ratios above 0.23.24,25It is evident from Figure 11 that the permeability of the bilayer membrane decreases with increasing molar ratios of steroids. The stability of bilayer membranes is also found to be dependent upon the kinds of steroids (i.e., cholesterol < stigmasterol < stadiene < acetate). As this tendency is very consistent with the results indicated in Figures 4 and 6, which represent the difference in the cohesive force between DPPC and steroids, the permeability of a bilayer membrane seems to increase with decreasing cohesive force between DPPC and steroids. Microviscosity of Bilayer Membranes. We have investigated the relationship between the permeability and microviscosity of the bilayer membrane. Generally, fluorescence polarization is well-known to be correlated to microviscositynear fluorescent probe^,^^^^^ which is calculated using the Perin-Weber’s equation.28 Microviscosity is increased with increasing fluorescence polarization. It has been reported that fluorescence polarization of 2Ap in the bilayer membrane indicates the level of microviscosity near the hydrophilic group of the bilayer membrane.29 (24) Sunamoto, J.;Iwamoto, K. InLiposomes; Nojima, S.,Sunamoto, J., Inoue, K., Eds.; Nankodo: Tokyo, 1988; p 34. (25) Kano, M.; Imaidzumi K. Cholesterol; Sankyo Suppan: Tokyo; 1987:, D_54. - ~
(26) Pugh, E. L.; Bittman, R.; Fugler, L.; Kates, M. Chem. Phys. Lipids 1989,50,43. (27) Abe, M.; Goto, M.; Ogino, K. J . Jpn. Oil Chem. SOC.1990,39, 3871
(28) Yagi, K.; Sekine, T. Application ofFluorescence Spectrometry to Biochemistry Research; 1976; p 34. (29) Inoue, T.; Muraoka, Y.; Fukushima, K.; Shimozawa, R. Chem. Phys. Lipids 1988,46,107.
Figure 12. The relationship between permeability and microviscosity at 35 “C. (DPPC/steroid/DCP = 7:3:1): 0, stig0 ,stigmasterol. masterol acetate;014,22-stigmastadien-3-one;
The relationship between permeability (after a 72-h incubation) and the microviscosity of the bilayer membrane is shown in Figure 12. The permeability decreases effectively with a n increase in microviscosity near the hydrophobic groups in the following order: acetate > stadiene > stigmasterol. Our previous paperg reported that permeability increases with an increase in the fluidity of the bilayer membrane since “kink isomers’’ are susceptible of occurring. It is found that permeability of the bilayer membrane decreases with increasing microviscosity andor decreasing fluidity. Microviscosityof the bilayer membrane increases with increasing cohesive force between DPPC and steroids in the mixed monolayer in the following order: acetate < stadiene < stigmasterol. Therefore, it can be postulated that the permeability of the bilayer membrane decreases with increasing cohesiveforce between DPPC and steroids since the DPPC is not able to maintain its property as the bilayer membrane. Moreover, the limiting molecular area of the steroid alone in monolayers does not coincide with the tendency of the permeability of bilayer membranes in the following order: stigmasterol < acetate < stadiene. These results suggest that the hydrophilic groups in the steroids have greater influence on the bilayer membrane properties than the orientation of the steroids owing to steric hindrance effects.
Conclusion This study provides four conclusions: (1)The cohesive power between DPPC and the steroid molecules increases with decreases in the difference between the collapse pressure of DPPC alone and those of the steroids. (2) The molar ratio of DPPC to steroid to form liposomes is dependent upon types of hydrophilic groups in the steroids. (3) Liposomes cannot form for the mole ratios of steroid which give a two-stage collapse in a DPPCIsteroid mixed monolayer. (4) The permeability of a bilayer membrane decreases with the increase of cohesive forces between DPPC and steroids. Therefore, the stability of liposomes increases with the increase of cohesive power between DPPC and the steroids.
Acknowledgment. This research was supported by the Cosmetology Research Foundation, Japan. LA9404393
