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Langmuir 1997, 13, 3674-3680
Intercalation and Bilayer Formation of Phospholipids in Layered Synthetic Mica. 2.† Solvent Effect of the Intercalation Reaction of Natural and Reduced-Type Phosphatidylcholines Yasushi Kanzaki,* Midori Hayashi, Chie Minami, Yoshihiro Inoue, Makoto Kogure, Yoshiteru Watanabe, and Tatsuo Tanaka Showa College of Pharmaceutical Sciences, 3-3165, Higashi-Tamagawagakuen, Machida, Tokyo 194, Japan Received August 26, 1996. In Final Form: April 25, 1997X The intercalation reaction of natural phospholipids into synthetic mica was examined by means of chemical analysis and X-ray diffraction analysis. The rate of the intercalation was fast from a chloroform solution of lipids, and bilayer formation in the solid phase was perfect. On the other hand, the rate of intercalation was rather slow in ethanol solution, and bilayer formation was not always successful. Such a solvent effect was accountable due to the solvation of the hydrophilic quaternary ammonium head either in the solution phase or in the solid phase.
Introduction Natural phospholipids such as phosphatidylcholine and phosphatidylethanolamine, which have hydrophobic dual alkyl chains and a hydrophilic head, are known to form stable bilayers due to the weak van der Waals forces caused by the long alkyl chains. Such a bilayer forms a wall and surrounds a biological cell to form stable biological bodies in the life system. According to rather flexible chemical bonds between lipid molecules, the bilayer membranes can act as various selective membranes which are essential to the biological activity. If such selective membranes were immobilized stably, the biological functions would be widely utilized in various fields. From such a point of view, the authors have studied the bilayer formation of alkyl amines with various chain lengths and natural phospholipids in layered inorganic compounds, such as transition metal phosphates.1,2 Such reactions are known as the intercalation reaction. Then the synthetic procedure has been extended to the intercalation reaction of several biological compounds, e.g. enzymes3 and hemoglobin.4 Sodium-type synthetic mica (Na-TSM)5 is selected as the host layered compound in this study. Na-TSM is a perfectly neutral inorganic compound6 and can intercalate ethanol molecules easily and swell the interlayer. The reverse deintercalation occurs reversibly within a few minutes only in dry atmosphere. We can follow the reversible process directly using the X-ray diffractometer. The authors have reported the successful intercalation and bilayer formation of phosphatidylethanolamine using Na-TSM,7 which was unsuccessful when the transition metal phosphates were used as the host layered materials.2 In the preceding paper, the intercalation reaction of n-alkyl amines was found to have considerable solvent † X
Part 1: Kanzaki, et al. Langmuir 1993, 9, 1930. Abstract published in Advance ACS Abstracts, June 15, 1997.
(1) Kanzaki, Y.; Abe, M. Bull. Chem. Soc. Jpn. 1991, 64, 1846. (2) Kanzaki, Y.; Abe, M. Bull. Chem. Soc. Jpn. 1992, 65, 180. (3) Kanzaki, Y.; Abe, M. Bull. Chem. Soc. Jpn. 1991, 64, 2292. (4) Shimizu, G.; Kanzaki, Y.; Tanaka, T. Bull. Chem. Soc. Jpn., to be published. (5) Toraya, H.; Iwai, S.; Marumo, F.; Daimon, M.; Kondo, R. Z. Kristallogr. 1976, 144, 42. (6) Morikawa, Y.; Goto, T.; Moro-oka, Y.; Ikawa, Y. Chem. Lett. 1982, 1667. (7) Kanzaki, Y.; Kogure, M.; Sato, T.; Tanaka, T; Morikawa, Y. Langnuir 1993, 9, 1930.
S0743-7463(96)00837-2 CCC: $14.00
effects.1 Thus the solvent effect and the difference in natural phospholipids on the intercalation into Na-TSM are investigated in this study. Experimental Section Materials. Sodium-type synthetic mica (tetrasilicic fluoro mica (Na-TSM: Na[Mg2.5Si4O10F2]‚2H2O)5 used as a host inorganic layered compound was supplied from Topy Industry Co. (Japan). Phospholipids used as guest compounds are reducedtype natural phosphatidylcholine from soybeans, which was prepared by hydrogenating the residual double bonds [H-PC: Lecinol S-10EX (Nikko Chemicals Co. Ltd.), C18/C16 ) 85:15 (averaged ratio and averaged molecular weight, 786), iodine value less than 0.97], and lecithin from egg yolk [phosphatidylcholine: E-PC (Wako Pure Chemicals, Ind.)]. Chloroform (reagent grade) and ethanol (reagent grade) were used as solvents to dissolve each phospholipid without further purification. Preparation of the Intercalation Compound. Intercalation was carried out as follows. Powdered Na-TSM (0.1 g) was contacted with 10 cm3 of chloroform or ethanol solution containing a known amount of phospholipid (Na-TSM/lipid, 1.0:0.4 to 1.0: 2.0 in mole ratio). Temperature was varied from -30 to +37 °C and the reaction time from 0 to 7 days. A standard reaction condition in which a typical bilayer was successfully formed was temperature, +37 °C, reaction time, 72 h, Na-TSM/lipid ) 1.0: 0.8. This preparative condition is termed as “standard condition” in this study unless otherwise specified. Characterization. The product after contacting Na-TSM with lipid solution under given conditions was washed and filtered on a filter paper using a pure solvent. The amount of intercalated phospholipid was estimated by subtracting the residual amount of phospholipid in solution from the added amount of phospholipid. The former was determined gravimetrically after vaporizing the solvent. X-ray diffraction analysis (Cr KR radiation (λ ) 0.228 97 nm) equipped with a silicon single crystal monochromator) was carried out on the sample, which was developed on a glass plate. The lattice parameter of around 7 nm can be reliably determined using Cr KR without using any special technique such as the low-angle scattering method. Thermal analysis was carried out by means of TG (thermal gravimetry), DSC (differential scanning calorimetry), and DTA (differential thermal analysis). The heating rate was 10 °C/min, and TG-DTA or TG-DSC curves were measured in an atmosphere of nitrogen or air.
Results and Discussion Two kinds of phosphatidylcholines were selected as the guest compounds. One was the typical natural phosphatidylcholine extracted from egg yolk (E-PC) without © 1997 American Chemical Society
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Figure 1. Schematic structure of hydrogenated PC (H-PC) and as extracted PC (E-PC).
Figure 2. Structural view of sodium-type tetrasilicic fluoro mica (Na-TSM‚2H2O) (a) and conceptual view of lipid bilayer formation in layered inorganic materials (b).
further pretreatment. The lipid may contain a few double bonds in alkyl chains. The other is the reduced-type phosphatidylcholine (H-PC) prepared from soybeans in which most of the residual double bonds have been hydrogenated by using metal catalysis. H-PC must have straight alkyl chains since few double bonds existed in the alkyl chains. The schematic structures of both phosphoplipids are shown in Figure 1. The lengths of these molecules in Figure 1 were estimated from the d-spacing of their bilayers (See Figure 3). Generally speaking, the phosphatidylcholines, which have a quaternary ammonium head, can form a rather more stable bilayer structure than phosphatidylethanolamine,7 which has a simple ammonium head. According to the chemical analysis, these lipids were found to be intercalated in a neutral form (intramolecular ion pair: Figure 1), and no evidence for the ion exchange process was observed because the amount of the released Na+ ions was very small. General Feature of the Intercalation in Na-TSM. The products were first characterized by X-ray diffraction analysis. The outline of X-ray analysis of the host NaTSM is described in advance. Figure 2a shows the structural view of general clay minerals including NaTSM. All atoms are combined with strong covalent bonds in the rigid layers (solid lines), and sodium ions and water molecules are located between the rigid layers. The ions and molecules located between the interlayers are combined with the host rigid layers due to the weak hydrogen bonding and are rather mobile between the layers. The phospholipid bilayers can also be formed between the host rigid layers, as illustrated in Figure 2b. Corresponding X-ray diffraction patterns of Na-TSM containing two formal water molecules are shown in Figure 3a. (The dehydration was found to occur in two steps, as will be seen in the TG-DTA curve in Figure 7a. Each peak
corresponded to the formation of monohydrate and dehydrate states, respectively.) Strictly speaking, the crystal system of Na-TSM‚2H2O has been ascribed to a monoclinic lattice, space group of C2/m, with a ) 0.525 nm, b ) 0.9078 nm, c sin β ) 1.54 nm.5 In dehydrated Na-TSM, c sin β ) 0.95 and β was estimated to be about 100°. The base area of a unit cell is thus 0.477 () ab) nm2 (Z ) 2), which will be referred to a cross sectional area of alkyl chains in the bilayer structure of intercalated phospholipids (Table 3). These monoclinic structures can be approximated to a hexagonal structure if only the expansion of the d-spacing (i.e. c sin β) were discussed instead of the correct crystal structure. Thus, the d-spacings of the layered crystals are approximated to a hexagonal c-parameter in this paper. According to the approximation, the sequential peaks with an integral number of wave length (n in 2d sin θ ) nλ) can be described to be (001), (002), ..., which are typical of the layered materials with a large d-spacing. Figure 3a-e illustrates the X-ray diffraction patterns of Na-TSM‚2H2O, anhydrous Na-TSM, H-PC and E-PC bilayers, and the intercalation compound of E-PC. Monohydrate (Na-TSM‚H2O) has a d-spacing of about 1.2 nm in comparison with 1.54 nm for Na-TSM‚2H2O. The anhydrous Na-TSM has a d-spacing of 0.96 nm. Thus the thickness of the Na-TSM rigid layers was estimated to be 0.96 nm, as seen in Figure 2a. The value 0.96 nm was used instead of 1.54 nm to estimate the size of the gallery height for the intercalation compounds. The second characteristic view of the X-ray diffraction patterns of PCs’ bilayers is that the intensity order of the peak height does not correspond to n in (00n). For example, the (004) peak of the bilayer of E-PC was higher than that of (002) and (003). Such a situation was more pronounced in H-PC, in which (002) and (003) peaks were undetectably small in comparison with the intensity of
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Figure 3. X-ray diffraction patterns of Na-TSM‚2H2O (a), anhydrous Na-TSM (b), bilayer of H-PC (c), bilayer of E-PC (d), and E-PC intercalation compound with Na-TSM (e).
(004). The d-spacings of E-PC and H-PC bilayers were 5.16 and 6.17 nm, respectively. The d-spacing of the E-PC intercalation compounds of Na-TSM under standard condition (Na-TSM/PC ) 1.0: 0.8 in mole ratio) which was prepared in the chloroform solution was 5.54 nm. The gallery height for E-PC molecules in this intercalation compound was estimated to be 4.58 nm ()5.54 nm - 0.96 nm), which is 0.58 nm narrower than the d-spacing of E-PC bilayers. The finding may indicate that the bilayers of E-PC were definitely formed between the host TSM layers and the alkyl chains must be inclined to some extent. On the other hand, the intercalation of H-PC prepared in the chloroform solution gave a different behavior. The d-spacing of the intercalation compounds of H-PC was 4.56 nm. The gallery height of this intercalation compound can be estimated to be 3.60 nm ()4.56 nm - 0.96 nm) in comparison with 6.17 nm for H-PC bilayers. Thus the formation of H-PC bilayer must be unsuccessful between the host TSM layers. The gallery height was 0.51 nm larger than the thickness of the hypothetical monolayer of H-PC, i.e. 3.09 nm ()(6.17/2) nm). The detailed results on d-spacing will be shown below. It should be noted that overstrict reliance on the d-spacing seems speculative because the reproducibility of observed d-spacings in X-ray diffraction patterns was rather poor, 0.2 nm or so depending on the preparative condition or drying condition. Temperature Dependence of the Intercalation Reaction. The intercalation of phospholipids was found to have a strong temperature dependence. Figure 4a-d shows the X-ray diffraction patterns of the reaction products of H-PC prepared between -30 and +37 °C. Almost no intercalation occurred at -30 °C. The reaction proceeded very slowly at +3 °C. As the temperature increased, the X-ray diffraction pattern began to indicate
Kanzaki et al.
Figure 4. Temperature dependence of X-ray diffraction pattern of H-PC intercalation compound with Na-TSM: Na-TSM‚2H2O (a) and prepared in chloroform solution at -30 °C (b), -3 °C (c), and +37 °C (d). Na-TSM/H-PC ) 1.0:0.8 in mole ratio. Reaction time: 3 days.
the formation of a single phase, and a complete singlephase product appeared at +37 °C. The temperature dependence of E-PC intercalation was similar to that of H-PC. The temperature dependence was similar in chloroform and ethanol solutions. Solvent Effect on the Intercalation Reaction. It has already been reported that the intercalation of n-alkyl amines into inorganic layered compounds such as γ-titanium and γ-zirconium phosphates had a strong solvent effect either in the reaction rate or in the reaction product.1 A similar effect is also anticipated in natural phospholipids. Figure 5a,c shows the X-ray diffraction patterns of reaction products at a reaction time of 7 days. Table 1 summarizes the correspond change in the d-spacing at various reaction times. The appearance of a single-phase product is the measure of the reaction rate. It was found that the reaction rate was considerably faster in the chloroform solution than in the ethanol solution. In addition, a near single phase product was formed just after the contact of Na-TSM with H-PC solution of chloroform. The rate in ethanol solution was very slow, and a single-phase product appeared after 24 h in H-PC and 72 h in E-PC. Figure 6a,b indicates the time dependence of the amount of H-PC and E-PC incorporated in the host Na-TSM layers observed in chloroform and in ethanol solutions. Both results show that the rate of intercalation was appreciably faster in chloroform solution than in ethanol solution, although the saturated values were not so different. The role of the solvent in the case of intercalation reactions is still ambiguous. The solvent effect, however, must be superior in the solution phase to that in the solid phase, because the lipids must be fully solvated in the solution phase and the amount of the cointercalated solvent molecules was expected to be small. (The role of the incorporated solvent molecules will be discussed in
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Figure 5. X-ray diffraction pattern of H-PC intercalation compound with Na-TSM prepared at Na-TSM/H-PC ) 1.0:0.8 in chloroform (a) and in ethanol (c). Temp: 37 °C. Reaction time: 7 days. (See also Table 1.) X-ray diffraction pattern of H-PC intercalation compound prepared at Na-TSM/H-PC ) 1.0:2.0 in chloroform (b) and in ethanol (d). Temp: 37 °C. Reaction time: 3 days. (See also Table 2.)
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Table 1. Time Dependence of d-Spacing (in nm) and Phase Appearance Na-TSM/H-PC ) 1.0:0.8; Temp 37 °C mixed phase d/nm (in chloroform)
single phase
0h
2h
6h
24 h
72 h
168 h
1.48 4.50
1.47 4.36
1.47 4.52
4.40
4.46
4.53
mixed phase d/nm (in ethanol)
single phase
0h
2h
6h
24 h
72 h
168 h
1.37
1.37 4.45
1.36 4.42
1.47 4.38
1.47 4.41
4.39
the Thermal Analysis section again.) Since the energy of the hydrophobic interaction in polar media is generally a few orders lower than that of the hydrophilic interaction, the solvent effect observed here was ascribable mainly to the solvation of the polar head. Thus the solvent effect must be stronger in ethanol than in chloroform solution, as the former molecule has a larger dipole moment than the latter. The desolvation process from the polar head upon insertion of phospholipid molecules must control the rate of the intercalation. Concentration Dependence on the Intercalation Reaction. It is important to know how many phospholipid molecules are intercalated into the host layered compound. Figure 5b,d shows the X-ray diffraction patterns of the H-PC intercalation compounds prepared in chloroform and ethanol solutions, respectively, at NaTSM/H-PC ) 1.0:2.0. Corresponding concentration dependence of d-spacing is summarized in Table 2. In the case of chloroform solution, a single-phase X-ray diffraction pattern was once observed at the concentration range around Na-TSM/H-PC ) 1.0:0.8, and the d-spacing was around 4.56 nm where the composition of the product was estimated to be about Na-TSM/H-PC ) 1.0:0.3 (See Figure 6). At higher concentration range two phases appeared. A well-defined single-phase X-ray diffraction pattern was observed again at a concentration around Na-TSM/H-PC ) 1.0:2.0. The d-spacing of this product was estimated to be 7.23 nm. The gallery height for the lipid molecules was calculated to be 6.27 nm (7.23 - 0.96) which was about the same size as that of the H-PC bilayers, 6.17 nm. The finding indicates that the bilayers of H-PC were definitely formed in the host layered material in a manner similar to that of the H-PC bilayer (Figure 3c). The product prepared in ethanol solution also gave a single-phase X-ray diffraction pattern at all concentration ranges observed. The maximum d-spacing observed in ethanol solution, 4.60 nm, was too small in comparison with the thickness of the H-PC bilayer, 6.17 nm. It can be concluded that the bilayer formation of H-PC was unsuccessful in the host lattice as long as ethanol was used as a solvent. In the case of E-PC, the intercalation reaction was similar in chloroform and in ethanol solutions. The maximum d-spacings were 5.32 and 5.54 nm, respectively, at a concentration of Na-TSM/E-PC ) 1.0:2.0. These values may indicate the formation of more inclined bilayer in the host layered compound than in the pure E-PC bilayer. Thermal Analysis. Figure 7a-d shows the thermal analysis data of Na-TSM containing two formal water molecules (a), the bilayer form of H-PC (b), and two H-PC intercalation compounds with different d-spacings ((c) 4.56 nm, (d) 7.23 nm). Two endothermic peaks at 87.8 and 142.7 °C for Na-TSM accompanying a large weight loss correspond to the stepwise desorption of two formal water molecules. These endothermic peaks disappeared completely in the H-PC intercalation compounds (c, d) and suggested the removal of water molecules during the
Figure 6. Time dependence of amount of incorporated H-PC (a) and E-PC (b) observed in chloroform solution (0) and ethanol solution (b). Na-TSM/H-PC ) 1.0:0.8. Temp: 37 °C.
intercalation process. Figure 7b shows the TG-DTA curves of the bilayer form of H-PC prepared by developing the chloroform solution in which a simple sharp endothermic peak at 95.4 °C was observed and indicated the phase transition from hexagonal to liquid crystal form. Two kinds of H-PC intercalation compounds with different d-spacings showed a similar sharp endothermic peak. The intercalation compounds with a d-spacing of 4.56 nm, where a monolayer like H-PC was thought to be formed (see Table 2), showed an endothermic peak at 65.1 °C, while that with 7.23 nm showed a peak at 63.4 °C, and the difference was rather small. The endothermic peak of the H-PC intercalation compound with a d-spacing of 4.34 nm which was prepared in ethanol solution appeared at a lower temperature, 59.7 °C. The difference in the phase transition temperature was unexpectedly large between the simple H-PC bilayer and the intercalation compounds. This lower shift observed in the intercalation compounds resembles the lower shift of the PC-water mixture.8 The lower shift observed in the PC-water mixture has been attributed to the separation of neighboring bilayer sheets due to the insertion of water molecules between hydrophilic head layers. In the present case, the separation of the lipid bilayer sheet was achieved by the host inorganic layer (Figure 2b). The large lowtemperature shift observed here may be attributed to the strong interaction between the hydrophilic head and hydrophilic inorganic surface. In contrast to the sharp endothermic peak which was independent of weight loss, a very slow weight loss was observed corresponding to the wide endothermic peak of (8) Chapman, D.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, 1, 445.
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Table 2. Concentration Dependence of d-Spacing (in nm) and Phase Appearance: Temp 37 °C; Reaction Time 3 Days mixed phase
d/nm (in chloroform)
single phase
mixed phase
0 H-PC/TSM
0.4 H-PC/TSM
0.8 H-PC/TSM
1.0 H-PC/TSM
1.5 H-PC/TSM
single phase 2.0 H-PC/TSM
1.44
1.53 5.07
4.56
4.72 7.12
7.15
7.23
single phase
d/nm (in ethanol)
0 H-PC/TSM
0.8 H-PC/TSM
1.2 H-PC/TSM
2.0 H-PC/TSM
1.44
4.34
4.45
4.60
Figure 7. Thermal analysis curve (TG-DTA or TG-DSC) of Na-TSM‚2H2O (a), bilayer of H-PC (b), and H-PC intercalation compound prepared at Na-TSM/H-PC ) 1:0.8 (c) and Na-TSM/H-PC ) 1:2.0 (d). Sample weight of a, c, d 10 mg.
the DTA curve and indicated the desorption of solvent molecules. If all weight loss up to 100 °C was ascribable solely to the vaporization of the solvent molecules, the amount of the solvent molecules cointercalated can be estimated to be 3.0 and 2.9 wt % for ethanol and chloroform, respectively. The amounts roughly correspond to 1:0.5 and 1:0.3 (H-PC/solvent molecule) in mole ratio. The amount of cointercalated solvent molecules was found to decrease with increasing the amount of intercalated lipid molecules, 6% (reaction time 3 days) to 3% (reaction time 7 days) in the sample prepared in the ethanol solution. The difference was small in the case of chloroform solution. This finding suggests that the solvent effect might also exist in the solid phase. Bilayer Form of Lipid in Intercalation Compounds. The amount of incorporated phospholipids was estimated gravimetrically from the difference between the added amount of phospholipids and the residual amount in the solution. In this case, the result was very sensitive to the washing process. Thus the observed amount of phospholipids shown in Figure 6a,b might be overestimated especially at a high concentration region. The incorporated amounts of phospholipids were also calculated from the weight loss in the TG curve for comparison since a relatively flat plateau was observed above 380 °C in the TG curve. The two results are plotted in Figure 8. Since the decomposition product was not analyzed chemically, the calculated values based on the TG curve must be somewhat underestimated. The true value must lie between the two curves. The average area of the cross section of each alkyl chain was calculated assuming that the phospholipid molecules formed bilayers and were intercalated just perpendicular
Figure 8. Concentration dependence of amount of incorporated H-PC: (4) value calculated from the residual amount of H-PC and (2) calculated from the TG curve.
to the host layer surface. The second assumption also indicated that the bilayer of lipids was formed in each TSM interlayer homogeneously. According to these two assumptions, the area that was occupied by each alkyl chain (two chains per molecule) was calculated. The calculation was carried out on three intercalation compounds of PC. Sample 1 was prepared in chloroform solution at Na-TSM/H-PC ) 1.0:0.8 having a d-spacing of 4.56 nm, sample 2 was prepared in chloroform solution at Na-TSM/H-PC: ) 1.0:2.0 having a d-spacing of 7.23 nm, and sample 3 was prepared in chloroform solution at
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Table 3. Occupied Cross Sectional Area per Alkyl Chain for Various Intercalation Compounds; Base Area Was Estimated from Crystallographic a- and b-Parameters
cross sectional area per chain (nm2) a
base area (bilayer)
closely packed (theoretical)
0.48a
0.24
Na-TSM/H-PC 1.0:0.8 (d ) 4.56) 1.0:2.0 (d ) 7.23) 0.71
0.37
Na-TSM/E-PC 1.0:0.8 (d ) 5.54) 0.57
a(0.525) × b(0.9078).
Na-TSM/E-PC ) 1.0:0.8 having a d-spacing of 5.54 nm. The results are summarized in Table 3. The base area of the host Na-TSM was estimated to be 0.48 nm2 per two formula weight. Thus each lipid molecule having two alkyl chains can occupy 0.48 nm2 if the composition of Na-TSM/H-PC ) 1.0:1.0 and can form a bilayer in each gallery space. On the other hand, the cross sectional area of the closely packed alkyl chain that was calculated for the n-alkyl amine intercalation compound of R-zirconium phosphate9 is shown in Table 3 for comparison. In sample 2, in which almost perfect bilayers were assumed to be formed, the cross sectional area was estimated to be 0.37 nm2/chain, which is a little larger than that of the closely packed alkyl amine bilayers, 0.24 nm2/chain. The calculated cross sectional area was 0.71 nm2/chain for sample 1, which is twice as large as that estimated in sample 2. Since the bilayer formation was unsuccessful in this sample, the large value must suggest the formation of the monolayer. If this is true, the cross section was estimated to be 0.36 nm2/chain. The cross sectional area of sample 3, in which E-PC was intercalated was calculated to be 0.57 nm2/chain, and the value seems too large to form a stable bilayer. If alkyl chains of E-PC were inclined largely, the inclination must result in the apparent large cross section. In fact, the apparent cross (9) Costantino, U. J. Inorg. Nucl. Chem. 1981, 43, 1895.
section decreased to 0.40 nm2/chain if alkyl chains were inclined 45° against the host layer surface. In addition, the value of 0.57 nm2/chain can reasonably be ascribed to the inclined bilayer formation if the presence of double bonds in E-PC alkyl chains (Figure 1b) were taken into account. Conclusion. The phospholipid bilayers were successfully formed in each interlayer of the inorganic layered compound according to the use of synthetic Na-TSM. They are very stable, and the immobilized bilayers will be applicable to various biological means such as support of unstable medicines. Further application of these compounds to the drag delivery system (DDS) will be published soon.10 Acknowledgment. This study was financially supported by the grant of the Japan Private School Promotion Foundation and also the cooperation research program of Showa College of Pharmaceutical Sciences. The authors wish to thank Prof. Y. Morikawa, Tokyo Institute of Technology, for his helpful suggestions and discussion, and Mr. Sugimori, Topy Industry, for suggestions on TSM. LA9608371 (10) Kanzaki, Y.; Okano, M.; Tsukamoto, M.; Uchida, F.; Shimoyama, Y.; Inoue, Y.; Kogure, M.; Watanabe, Y. Chem. Pharm. Bull., in preparation.
