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Anal. Chem. 1991, 63,203-207
Absorption Behaviors of Surfactant Molecules on a Lipid-Coated Quartz-Crystal Microbalance. An Alternative to Eye-Irritant Tests Yoshio Okahata* a n d Hiroshi Ebato Department of Biomolecular Engineering, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, J a p a n
The absorption behaviors of 13 kinds of surfactant molecules In a lipid matrlx were studied by using a synthetic dlmethyidloctadecylammonlum poly( styrene-Csulfonate) (2C1,N+2C1/PSS-) microbelance or a naturally occurring dipabnltoylphosphatldyiethanolamlne(DPPE) multlbliayer filmcoated quartz-crystal microbalance (QCM) in distilled water. The resonance frequency of the lipldtoated QCM decreased ilnearly wlth increasing the mass of substances deposited in the lipid matrix on the QCM, and the partltion coeffklents (P) and dnludon rate constants ( D ) were obtalned quantltatlvely. There exlsted a good correiatlon between the log P values of the 13 surfactants in the lipid matrix on the OCM and the eye-irritant value (the Dralze score) obtained from animal experlments. the surfactant molecule showing the hlgher Draize score (higher eye lrrltancy of rabblts) gave the larger partltlon to the llpid matrix. Slgnmcant correlations were not observed between the Dralze score and partltlon coefflclents In protein layers on the QCY or the hydrophilic-Iipophillc balance (HLB) of surfactants. Thus, the eye Irritancy could be expialned by the absorptlon behavlors In the lipid matrix of the biological membranes, but not by the slmpie hydrophoblclty. P and D values increased drastically at the phase transition ( T , ) from the solid state to the liquid-crystalline state of the 2C1,,N+2C1/PSS- multlbliayer film. Surfactant molecules absorbed and penetrated deeply Into the lipid matrix as a monomerlc form but not an aggregated mkelar form.
In the technique of Draize et al. (1) used to predict eyeirritant potential, surfactants or chemical substances were instilled into rabbit eyes in vivo and irritation was assessed by numerical scoring. Recently, the predictive reliability of this technique has been questioned (2-4) and its use of living animals has been criticized. This test requires skillful techniques of objective judgement and a large number of animals must be used. Consequently, a simple and more reliable in vitro test has been sought to replace the animal test. Borenfreund and Shopsis have reported that both the uptake of radioactive uridine by cells and the morphological change of cells observed by light microscopy in a mammalian cell culture are closely related to the results of the Draize test (5). Kemp has developed a methodology to evaluate the toxicity of surfactants to mouse fibroblast cells from the coloration of the cultured cells arising from the damage to the cell caused by the surfactants (6). Sunamoto et al. reported that the physicochemical lysis of liposome membranes by surfactants, followed by dye release from the inner aqueous phase, was correlated with the results of the Draize test with the same surfactants (7).Igarashi et al. measured the turbidity of intact cornea after treatments with surfactants by using an opacitometer and found a correlation with the Draize score (8).
*Towhom all correspondence should be addressed. 0003-2700/91/0363-0203$02.50/0
In this paper, we quantitatively obtained the partition coefficients of various surfactants in the lipid multilayer matrix by means of a synthetic lipid-coated quartz-crystal microbalance (QCM). QCMs are known to provide mass-measuring devices in nanogram levels, because the resonance frequency changes sharply upon the deposition of a given mass on the electrode (9). The simple synthetic multibilayer-immobilized film, dimethyldioctadecylammonium poly(styrene-4-sulfonate) (2C18NC2C1/PSS-), and the phospholipid, dipalmitoylphosphatidylethanolamine (DPPE),cast film were used as a lipid matrix on a QCM (10-15). The experimental apparatus is shown in Figure 1. Partition coefficients of 13 surfactants in the lipid matrix obtained from the frequency changes of the lipid-coated QCM were linearly correlated with the Draize score for the same surfactants. The surfactant having the stronger eye irritancy (the higher Draize score) showed the higher partition in the lipid matrix on the QCM. The procedure is a simple and reusable one for the screening of potential irritants and is also applicable as a new device to study the interaction of surfactant molecules with the lipid matrix of the cell membrane. EXPERIMENTAL SECTION Materials. Preparations of a polyion complex of 2ClsN+2Cl/PSS- were reported elsewhere (16-18). Surfactant molecules and DPPE were commercially available and used without further purification. Lipid-Coated QCM. A quartz-crystal microbalance (8-mm diameter, AT cut, 9 MHz) was connected to a homemade oscillator designed to drive the quartz at its resonance frequency in aqueous solution (10-15). The QCM was driven at 5-V dc, and the frequency of the vibrating quartz was measured by a frequency counter (Iwatsu Co., Model SC7201) attached to the microcomputer system (NEC Co., Model PC 9801) (see Figure 1). The following equation has been obtained for the AT-cut shear mode QCM:
where A F is the measured frequency shift (Hz), Fothe parent frequency of QCM (9 x loe Hz),Am the mass change (g), A the electrode area (0.20 cm2),pq the density of quartz (2.65 g cm-?, and the shear modulus (2.95 X 10" dyn cm-2). Calibration of the Q8M used in our experiments showed that a frequencychange of 1 Hz corresponds to a mass increase of 1.05 0.01 ng on the electrode of the QCM (10-15): Am = -[(1.05 f 0.01) X 104]AF (2)
*
A chloroform solution of 2ClsN+2C,/PSS- or DPPE was cast on electrodes on both sides of the QCM, dried in air, and aged in water at 60 "C for 1h. X-ray diffraction analyses showed that
2C1$J+2C1amphiphiles form extended lamellar structures of lipid bilayer (3.8 nm thick) parallel to the film plane (the QCM plate) in the polyion complex with poly(styrene-4-sulfonate)anions (14-17). The bilayer film showed a sharp endothermic peak at 45 "C with differential scanning calorimetry (DSC) in aqueous solution, corresponding to the phase transition from the solid to the liquid-crystallinestate (14,17). Similar multibilayer structures 0 1991 American Chemical Society
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P Personal computer
Frequency counter
Oscillating circuit
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I
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Surfactants
Multibilayer Film-immobilized Quartz-Crystal Microbalance
1
0
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Time/min. Figure 2. Frequency changes of the 2C,8N+C1/PSS- mukibilayer film (10 pg) coated QCM responding to additions of (a) hexadecylpyridinium chloride (40 ppm, 1.4 X M) and (b) sodium dodecyl sulfate (40 ppm, 1.2 X l o 4 M) at 45 "C. An aqueous solution of surfactants was i n w e d into distilled water at the closed arrow and the aqueous solution was changed to distilled water at the open arrow.
N
'Ooo
: 500
Y-
Figure 1. Experimental apparatus for frequency measurements of a lipid-multibilayer-coated quartz-crystal microbalance (QCM).
and the phase transition (T,= 52 "C) were observed in the DPPE cast film on the QCM. When the lipid film was cast 10 i 0.1 pg on the electrodes (20 mm2 X 2) on both sides of the QCM, the vibration decreased 10500 i 100 Hz in the air, which was consistent with the mass deposited on the electrode in line with eq 2. The lipid-film-coated QCM was soaked in 10 mL of distilled water, and an aqueous solution (5-50 pL) of surfactants was injected with stirring (see Figure 1). Frequency changes of the QCM were followed with time due to the absorption of surfactant molecules in the lipid matrix on the QCM. Each experiment was carried out three times and the average contained within an experimental error of &10 Hz. The eye-irritancy tests were carried out in Shiseido Basic Research Laboratories according to the established technique by Draize (I, 1.9) by using female Japanese albino rabbits weighing 2.5-3.0 kg. Into the conjunctiva sac of the right eyes of the rabbits was instilled 0.1 mL of 10% aqueous solution of surfactants. The left-hand eye remained untreated to serve as the standard for comparison. A t 24 h after the instillation, ocular changes about the cornea were scored according to the standard method (20).
RESULTS AND DISCUSSION Absorption Behaviors of Surfactants. Figure 2 shows typical frequency changes of the 2CI8N+2C1/PSS-multibilayer film (10 pg, 0.5 pm thick) coated QCM responding to additions of hexadecylpyridinium chloride and sodium dodecyl sulfate into 10 mL of distilled water a t 45 "C. When 40 ppm (1.2 X M) hexadecylpyridinium chloride was added, the frequency immediately decreased (LW = 510 f 10 Hz) and reached equilibrium within 1min, which corresponds to the absorption of 565 f 10 ng in the lipid matrix on the QCM from eq 2. The frequency reverted to the original value when the QCM was moved into new distilled water (at the open arrow in Figure l),which means the removal of absorbed surfactants from the lipid matrix to the aqueous solution was completed. These reversible absorption and desorption phenomena were able to be repeated several times without damaging the membrane and were observed for other surfactants. Partition
LA 50 60 5 40
0
T i m e / m i n. Figure 3. Frequency changes of the DPPE cast film-coated QCM responding to additions of hexadecylpyridiniumchloride [(a) 10 ppm, 3.7 X M; (b) 40 ppm, 1.4 X lo-' M] into distilled water at 25 "C.
coefficients ( P ) of surfactants in the lipid matrix from the aqueous phase were obtained in the following way. The concentration (moles/liter) of the surfactant taken up in the lipid matrix was divided by the concentration (moles/liter) of surfactants in the aqueous phase. The absorption amount (partition coefficients)of dodecyl sulfate was smaller than that of hexadecylpyridinium and the absorption behaviors changed largely depending on the chemical structure of the surfactants. The time courses of the frequency decrease in Figure 2 show penetration or a diffuse process of substances into the lipid matrix. The diffusion rate constant (D) can be calculated from the slope of a plot of AmJAm- versus t1I2for the initial absorption step, according to the approximations of the Hill and MacBain equation (21):
where Amt and Am- are the absorption amount a t time t and a t equilibrium, respectively. L is the membrane thickness. Figure 3 shows typical frequency changes responding to the addition of hexadecylpyridinium surfactants (10 and 40 ppm) when DPPE phospholipid cast films were employed as a lipid matrix on the QCM a t 25 "C. When 40 ppm surfactants was added in the aqueous solution, the frequency decreased a t the first stage and then increased slowly beyond the original value
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50 40 50
40
E -
30
?!
20
m
0
8
cn
-8 10 0
2.0
2.5
30
Relationship between the hydrophllic-lipophilic balance (HLB) of surfactants and the Draize score (cornea). The small HLB value indicates the large hydrophobicity. Numbers are the same as in Figure Flgure 5.
3.0
log P
4.
50 I
-9
-8
- 7
log ( D / c m 2 s-') Figure 4. Relationship between (a) partition coefficients, P , or (b) diffusion rate constants, D , of surfactants in the 2Cl,N+2Cl/PSSmultibilayer film-coated QCM and the Draize score (cornea)obtained for the same surfactants from animal experiments. 1, ClBH3,2, C18H3,N+(CH3),CI-; 3, Cl2H2,N+(CH3),CI-; 4, C13H2,CON(CH,)C4H4S03-Na+; 5, CI,H,,0S03-Na+; 6, Cl,H,5N+(CH,),CH,COO-Na ; 7, C8H1,(CBH,)@CH,),,OH; 8, sodium alkyl glyce I
(C,H,N+)CI-;
sulfate; 9, C1,H2,C0NHCH((CH2),C00-)C00-2Na+; 10, (C,H,),N 11, RCONH(CH,),N+(CH,),CH,COO-Na+;
r-
12, C8H,,(C8H4)(O-
sodium caseinate. to reach equilibrium (AF = 1200 f 20 Hz) after 1 h. This frequency enhancement from the original value is consistent with the mass (1300 f 30 ng) of the DPPE cast film on the QCM. When the small amount (2-10 ppm) of surfactants was added into the aqueous solution, the simple absorption behavior was observed as well as that in the 2C18N+2C1/PSSfilm in Figure 2. Thus, when the relatively large amount of surfactants was absorbed into the DPPE lipid matrix, the surfactants taken up will solubilize or cause the unstable lipid matrix to flake slowly into the aqueous phase from the plate even at room temperature. In the case of polyion complex 2CI8N+2C1/PSS-film, the flaking behavior was not observed even with the addition of a high concentration (ca. 400 ppm) of surfactants and a t high temperatures. This is why we employed mainly the synthetic and polymeric-type 2C18N+2C1/PSS- multibilayer cast film on the QCM in the following experiments instead of the monomeric phospholipid cast film. Correlations with the Draize Score. Absorption experiments were carried out on 13 surfactants (4 cationic, 5 anionic, 2 zwitterionic, and 2 nonionic amphiphilic molecules) 13,
20
HLB
0
(CH,),CI-; CH,),oOH;
10
by using the 2C18N+2C1/PSS-film-coated QCM in distilled water a t 45 "C. The amounts taken up to the lipid film on the QCM increased linearly with increasing the concentration of these surfactants in the aqueous solution in the range 104-10-4 M. The partition coefficients of the surfactants in the 2C18N+2C1/PSS- film were calculated from the slope of the plots of frequency changes versus concentrations. The diffusion rate constants (D) of surfactant molecules in the lipid matrix were also obtained from the initial time course of absorptions according to eq 3. The P and D values obtained by using the QCM method were plotted against the Draize score (cornea) for the same surfactants in Figure 4. The Drake score was obtained from the eye-irritancy test of rabbits according to the established standard method (1, 19, 20). There existed a good correlation ( r = 0.95) between log P of various surfactants in the 2C18N+2C1/PSS-film on the QCM and the Draize score (cornea) for the same surfactants (Figure 4a). The plot of log D versus the Draize score did not give a significant correlation (Figure 4b). The surfactant having the larger Draize score (the stronger eye irritancy) showed the higher partition toward the lipid matrix independent of the hydrophilic charge of surfactants. Thus, the intensity of eye irritancy seems to be determined by the absorption amount (but not by the diffusion rate constant) to the lipid matrix. The cationic single-chain surfactants showed high eye irritancy and a large partition to the lipid matrix; on the contrary, a sonicated cationic dialkyl surfactant (dioctadecyldimethylammonium salts) is hardly taken up in the lipid matrix and showed a very small Draize score. Since sonicated dialkyl amphiphiles are known to form lipid bilayer vesicles in aqueous solutions as well as phospholipids (22),a large vesicular aggregate seems to be hardly absorbed into the lipid matrix. The DPPE cast film was stable on the QCM only in the low concentration (2-10 ppm) of surfactants in the aqueous phase (Figure 3). Partition coefficients of 13 surfactants in the DPPE-coated QCM were obtained at the low concentration of surfactants in distilled water. A similar good correlation ( r = 0.92) between log P and the Draize score was observed for DPPE phospholipid membranes (not shown in the Figure). This means that the absorption behavior hardly depends on the detailed structure of the hydrophilic part of the lipid matrix, but the dialkyl bilayer structure is important for the absorption step. When the Draize score was plotted against the hydrophilic-lipophilic balance (HLB) of the same surfactants, a significant correlation was not observed (Figure 5). The HLB values are calculated from the comparison between the lipophilicity of an alkyl-chain part and the hydrophilicity of a head
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r
Cast amountiwg
I
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ii
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2
4
6
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10
. oi
1
10.6 5
600
2 400
7
1
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.
4
Membrane thickness1 pm
Flgure 7.
Effect of the thickness of the 2C18N+2C1/PSS-film on the
QCM on the absorption amount (Am) of hexadecylpyridinium chloride at 45 O C . -3
-2
-1
0
4 120
log ([surfactant]/ w i o h ) Figure 6. Relationship between the concentration of surfactants to cause a 50% release of fluorescent probes from liposomes and the Draize score (cornea)obtained from animal experiments. Numbers
are the same as in Figure 4. group in a surfactant, and the smaller HLB indicates the larger hydrophobicity of the surfactant (23). The result indicates that the eye irritancy is not simply explained by the hydrophobicity of surfactant molecules. Partition coefficients of various surfactant molecules for protein matrices were also obtained by using a kelatin-coated QCM at 45 OC. Kelatin is a protein existing in the skin and was chosen as a model of simple protein matrices because of the physical stability on the crystal plate at various conditions in aqueous solutions. The plot between log P values and the Draize score (cornea) did not give a significant correlation (not shown in the figure). The partition coefficients of surfactant molecules in a synthetic hydrophobic polymer matrix polystyrene-coated QCM did not show a significant correlation with the Draize score (not shown). These results indicate that the eye-irritant potency is determined mainly by the absorption behaviors into the lipid matrix but not proteins of biological membranes and is not explained by the simple hydrophobicity of surfactant molecules. Liposomes (lipid bilayer vesicles) have been available as a tool to investigate the pharmacological activity and the toxicity of physiologically active substances at the cell membrane level. Sunamoto and co-workers suggested a liposomal system (release experiments of carboxyfluorescein, CF) for the preliminary evaluation of the eye irritancy of surfactants (7). They observed a good correlation between the Draize score and percent CF release from the polysaccharide-coated liposomes for the addition of six kinds of surfactants. Kat0 et al. have also examined the release experiments of fluorescent probes (4-methylumbelliferyl phosphate) from the inner aqueous phase of DPPC/DPPE liposomes responding to the addition of surfactants into the outer aqueous phase at pH 7.4 and 25 "C (20). Permeation behaviors of probes from the liposome were observed when 10 surfactants were added in various concentrations, and the minimum concentration of surfactants to cause the 50% release of fluorescent probe from the liposomes was obtained. Figure 6 shows the relation between the Draize score (cornea) for 10 surfactants and the concentration of the same 10 surfactants to cause the 50% release of fluorescent probes from liposomes. A relatively good correlation (r = 0.85) was observed and the surfactant showing the 50% release of fluorescent probes a t the lower concentration gave the higher Draize score (the higher eye irritancy). However, the correlation was not very good compared with the results in Figure 4a using the lipid-coated QCM methods ( r = 0.95). In liposome experiments, the origin of the probe release from liposomes caused by added surfactant molecules
/ 01 10 20 30
io
50
'
0
Temperature/"C Figure 8. Temperature dependences of the partition coefficients ( P ) and diffusion rate constants ( D )of hexadecylpyridinium chloride on the 2ClBN+2C,/PSS-film on the QCM. The T , and T,' values were obtained from DSC measurements in the absence and presence of surfactants in aqueous solution, respectively. is not clear: a collapse of liposomes, a disturbance of the membrane structure by the incorporated surfactants, or a subtraction of lipids from the membrane by Surfactants. In the QCM method, only the partition behaviors of surfactants in the lipid matrix are observed, which may give the relatively good correlation with the Draize score. Moreover, the procedure of the QCM method is a simple, reliable, and reusable one for the preliminary screening of potential irritants compared with the liposomal experiments. Absorption Mechanisms. Figure 7 shows the effect of the membrane thickness of the 2ClsN+2C1/PSS- multibilayer film on the QCM on the absorption amount of hexadecylpyridinium chloride. The incorporation amount increased linearly with increasing the membrane thickness. The similar absorption behaviors were observed for other surfactant molecules employed. Thus, the surfactant molecule absorbs and penetrates deeply into the lipid multibilayer film and the absorption amount increased with increasing the membrane thickness. The phase transition from the solid to the fluid liquidcrystalline state of dialkyl chains is one of the fundamental properties of both natural and synthetic lipid bilayer membranes. The partition coefficients and diffusion or penetration rate constants of hexadecylpyridinium chloride were measured at various temperatures below and above the phase-transition temperature (T, = 45 "C) of the 2C18N+2C,/PSS-multibilayer cast film on the QCM, and the results are shown in Figure 8. P values showed a maximum near 30 "C, and D values simply increased at temperatures above 30 "C, which is low compared to the phase-transition temperature ( T , = 45 "C) of the 2C18N+2Cl/PSS- multibilayer matrix. The lipid film showed the relatively broad endothermic peak near 30 "C (Ti) in the presence of an excess amount of surfactant molecules in DSC measurements, although the film has a T, of 45 "C
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the surfactant molecules can absorb into the lipid matrix as a monomeric form but not in the aggregated micellar form. This is consistent with the result that the vesicular-forming amphiphile, dioctadecyldimethylammonium chloride, hardly absorbed on the lipid matrix (see number 10 of Figure 2). Liposomes did not interact with the lipid matrix on the QCM in this condition.
CONCLUSIONS
[Surfactant]/M
Figure 9. Effects of concentrations of (a) hexadecylpyridinium chloride and (b)Triton X-100 in the aqueous phase on the absorption amount in the 2C,,N+2C1/PSS- film on the QCM at 25 OC. The critical micellar concentration (cmc) of surfactants was obtained by electrical conductance methods.
in the absence of surfactants. These results indicate that the T, value of the 2C18N+2C1/PSS-multibilayers decreases near 30 "C (T,') due to the penetration of surfactant molecules into the lipid matrix and disturbing the membrane structure. The surfactant molecules can absorb and penetrate largely into the disturbed membrane only near T,', at which the solid and liquid-crystalline matrices coexist in the membrane, compared with the solidified state below T,' and the homogenously fluid state above T,'.The penetration rate increased simply in the fluid liquid-crystalline state above T,' compared with the solid state below T l . When partition coefficients of 13 surfactants into the 2C18N+2C1/PSS-membrane at 25 "C (below T,) were measured and plotted against the Draize score for the same surfactants similar to that in Figure 4, a good linear correlation was not obtained (P = 0.54, not shown in the figure). This may indicate that partition behaviors of surfactants in the fluid or disturbed lipid layer near and above T,reflects the eye irritancy of surfactants, because the biological membrane as a fluid state containing cholesterol and proteins. We should consider the effect of the viscoelastic changes of the lipid membrane near and above T , on the frequency change of QCM absorption and experiments, because the fluidity and T, of the membrane were affected by the incorporation of surfactants. We have reported that the frequency of the layered membrane (lipid multibilayer, smectic liquid crystal, and LB film) coated QCM discontinuously increases responding to the change from the solid to the fluid state of the membrane near T, when the membrane thickness is large on the QCM (13,14). However, when the membrane was coated with a thin layer on the QCM (less than 0.5 pm thick or a 10-pg amount on the QCM in the case of the 2C18N+2C1/PSS-membrane), the frequency was not affected by the fluidity and viscoelastic changes of the membrane (14). In this absorption study, we employed a 0.4-1.0-pm-thick 2C18N+2CI/PSS-membrane-coated QCM and confirmed that the frequency was changed only by the mass increase on the QCM, but not by the fluidity change of the membrane on the QCM. Figure 9 shows the effect of the concentration of surfactants in the aqueous phase on the absorption amount in the 2C18N+2C1/PSS-film on the QCM at 25 OC. The absorption amount of cationic hexadecylpyridinium chloride and nonionic Triton X-100 increased linearly with increasing the concentration of surfactants in the range 10-6-10-4 M; however, it became saturated a t the higher concentration nearly above the respective critical micellar concentration (cmc) of each surfactant. Partition coefficients of surfactants were obtained in the linear region of Figure 9. These results indicate that
The synthetic lipid-coated quartz-crystal microbalance can easily detect absorption behaviors of surfactant molecules into the lipid matrix from frequency changes in aqueous solutions. Partition coefficients obtained from the QCM method showed a good correlation with the Draize score obtained from animal experiments. The lipid-coated QCM would be expected as a simple, reliable procedure for the preliminary screening of eye irritants and will be useful as a new alternative nonanimal test to the Draize test. The QCM method is also effective to study the interaction mechanism of surfactant molecules with the lipid matrix. The lipid-membrane-coated QCM has been applicable for other bioactive compounds such as bitter substances (%), odorants (24, =),anesthetics (26), and antibiotics (27), in which the absorption or penetration step is predominant for the first step of the interaction with the biological cell membrane.
LITERATURE CITED Draize, J. H.; Woodard, G.; Calvery, H. 0. J. fharm. (Antwerp) 1944, 82, 377. Kay, J. H.; Calandra, J. C. J. SOC. Cosmet. Chem. 1982, 73, 281. Weltman, A. S.; Sparber, S. B.; Jurtshuk, T. Toxicol. Appi. fharmacoi. 1965, 7 , 308. Beckley, J. H. Am. Perfumer Cosmet. 1985, 8 0 , 51. Chem. Eng. News 1982, Oct 11, 7. Chem. Ind. 1982, 4 , 918. Sunamoto, J.: Iwamoto, K.; Imokawa. G.; Tsuchiya. S. Chem. fham. Bull. 1987, 35, 2958. Igarashi, H.; Katsuta, Y.; Matsumoto, H. J. Toxicoi. Sci. 1989. 74, 91. Igarashi, H.; Katsuta, Y.; Kawasaki, T. Fragrance J. (in Japanese) 1989. 1 1 . 63 S&&brey,G. Z.fhys. 1959, 755, 206. Okahata, Y.; Ariga, K. J. Chem. Soc.,Chem. Commun. 1987. 1535. Okahata. Y.; Ariga, K. Langmuir 1989, 5, 1261. Okahata, Y.; Ariga, K. Thin Solid Films 1989, 778, 465. Okahata, Y.; Kimura, K.; Ariga, K. J. Am. Chem. Soc. 1989, 7 7 7 , 9190. Okahata. Y.; Ebato, H. Anal. Chem. 1989, 67, 2185. Okahata. Y.; Ebato, H.; Ye, X. J. Chem. Soc., Chem. Commun. 1988, 1037. Okahata, Y.; En-na, G. J. fhys. Chem. 1988, 92,4546. Okahata, Y.; Taguchi, K.; Seki, T. J. Chem. SOC.,Chem. Commun. 1985, 1122. Okahata, Y.; En-na. G.; Taguchi, K.; Seki, T. J. Am. Chem. Soc. 1985, 707, 5300. Draize, J. H. Application of the Society of Chemicals in Food, Drugs, and Cosmetics; Association of Food and Drug Officials, US.: Austin, TX. 1959: I) 46. Kaio, S-.f Ikgaki, H.; Chiyoda, I.; Hagino, S.; Kobayashi, T.; Fujiyama. Y.; Kakoki, H.; Yamaguchi, M. Toxic. Vitro 1988. 2, 125. Shibusawa, T. J. Dyers Colour 1979, 95, 175. Kunitake, T.; Okahata, Y. J . Am. Chem. Soc. 1977, 99,3860. Okahata, Y. Acc. Chem. Res. 1988, 79, 57. Griffin, W. C. J. SOC.Cosmet. Chemists 1954. 5 , 249. Becker, P. I n Nonionic Surfactants; Schick, M. J., Ed.; Marcel Dekker: New York, 1967; p 608. Okahata. Y.; Ebato, H.; Taguchi, K. J. Chem. Soc., Chem. Commun. 1987, 1363. Okahata, Y.; Ebato, H.; En-na, G. Anal. Chem. 1990, 62, 1431-1438. Okahata, Y.; Shimizu, 0. Langmuir 1987, 3 , 1171. Okahata, Y.; Shimizu, 0.; Ebato. H. Buli. Chem. SOC.Jpn. 1990, 62, 1431-1438. Okahata, Y.; Ebato, H. &sui to Sosei (in Japanese) 1989, 25, 203. Okahata, Y.; Ebato, H. J. Chem. Soc., ferkin Trans. 2, in press. Okahata, Y.; Ye, X.; Shimizu, A.; Ebato. H. Thin Solid Films 1989, 780, 51.
RECEIVED for review May 29,1990. Accepted November 2, 1990. This study was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
