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Surface Tensiometric Study of Multiple Complexation and Hemolysis by Mixed Surfactants and Cyclodextrins Noriaki Funasaki,* Mariko Ohigashi, Sakae Hada,† and Saburo Neya Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Received December 4, 1998. In Final Form: August 27, 1999 The suppression of hemolysis induced with 1.0 mM dodecyltrimethylammonium bromide (DTAB) or 1.6 mM 3-(dimethyldodecylammonio)-1-propanesulfonate (DDAPS) by R-, β-, and γ-cyclodextrins (CyDs) is determined as a function of CyD concentration at 310 K, and is correlated with the surface tension values of their solutions. These surface tension data allow us to estimate the 1:1, 1:2, and 2:1 binding constants of DTAB or DDAPS with these CyDs. The 2:1 binding constants of DTAB and DDAPS with γ-CyD are larger than their 1:1 binding constants. This cooperative binding of DTAB and DDAPS to γ-CyD is ascribed to the fact that the γ-CyD cavity has an adequate space to accommodate two alkyl chains. Both the capabilities of CyDs for hemolysis suppression and surface tension elevation are in the order R-CyD ≈ β-CyD > γ-CyD for 1.0 mM DTAB and 1.6 mM DDAPS. The suppression of DTAB- or DDAPS-induced hemolysis for all the CyDs can be quantitatively predicted from the observed surface tension data, regardless of the kind and concentration of CyD. All the CyDs can bind the surfactants more strongly than phospholipid and cholesterol in the erythrocyte membrane.
Introduction Cyclodextrins (CyDs) have toroidal structures of different sizes. One side of the torus contains primary hydroxyl groups, whereas the secondary groups are located on the other side. The toroidal structure has a hydrophilic surface resulting from the 2-, 3-, and 6-position hydroxyls, making them water soluble. The cavity is composed of the glucoside oxygens and methylene hydrogens, giving it a hydrophobic character.1-4 CyDs, therefore, can entrap hydrophobic compounds in their cavity. Because CyDs are practically nontoxic, they are added into pharmaceuticals and foods for stabilization of labile compounds and long-term protection of color, odor, and flavor.1 CyDs have potential pharmaceutical applications, though they have not been licensed extensively.5 CyDs have a hemolytic effect at high concentrations5,6 because they can extract cholesterol and phospholipid from erythrocyte membranes.1,5-9 At low CyD concentrations, however, they can alleviate the hemolytic effects and the bitter taste intensities of drugs.1,5,10,11 Generally, both the desired pharmacological effects and the unwanted side effects are elicited only by the uncomplexed drug molecules.1,5,10,11 * Author for correspondence. † Deceased in July, 1999. (1) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1988; Chapters 1 and 3. (2) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; SpringerVerlag: Berlin, 1978; Chapters 2 and 3. (3) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344. (4) Connors, K. A. Chem. Rev. 1997, 97, 1325. (5) Fro¨mming, K.-H.; Szejtli, J. Cyclodextrins in Pharmacy; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1994; Chapters 3, 6, and 10. (6) Ohtani, Y.; Irie, T.; Uekama, K.; Fukunaga, K.; Pitha, J. Eur. J. Biochem. 1989, 186, 17. (7) Debouzy, J. C.; Fauvelle, F.; Crouzy, S.; Girault, L.; Chapron, Y.; Goeschl, M.; Gadelle, A. J. Pharm. Sci. 1998, 87, 59. (8) Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98, 2045. (9) Ishikawa, S.; Neya, S.; Funasaki, N. J. Phys. Chem. B 1998, 102, 2502. (10) Funasaki, N.; Uemura, Y.; Hada, S.; Neya, S. J. Phys. Chem. 1996, 100, 16298. (11) Funasaki, N.; Ohigashi, M.; Hada, S.; Neya, S. Langmuir 1999, 15, 594 and references therein. Equations 10, 11, 13, and 14 therein contain some errors and should be read as eqs 4, 5, 7, and 8 in the present paper.
Recently, we have shown that drug-induced hemolysis is suppressed by β- and γ-CyDs and that the extent of hemolysis suppression can be predicted by the surface tension value for the mixed drug and CyD solution, regardless of the kind and concentration of CyD.11 However, this prediction did not apply to R-CyD. For the reason for this failure we suggested that R-CyD binds the drug more weakly than membrane phospholipid.11 The CyD inclusion of surfactants has been investigated by many methods, and their 1:1 binding constants have been determined. Although recent literature values for the 1:1 complexation agree reasonably with each other, those for the 2:1 and 1:2 complexations are still few and unreliable.12 Surfactants have been used for the formulation of pharmaceuticals, and induce shape deformation and hemolysis by penetration into erythrocyte membranes.13-15 This surfactant-induced hemolysis may be suppressed by CyDs, because the concentration of the free surfactant molecule will be reduced. Furthermore, because surfactants have chemical structures similar to phospholipids, studies on the interaction between surfactants and CyDs serve to understand the hemolytic effect of CyD. The first purpose of this work is to demonstrate that we can quantitatively predict the extent of suppression of surfactant-induced hemolysis by R-, β-, and γ-CyDs from observed surface tensions. Because R-CyD can generally bind surfactants strongly,12 we can expect that the above prediction holds true even for R-CyD. The second purpose is to determine the 1:1, 1:2, and 2:1 binding constants of surfactant and CyD by the surface tension method10-12,16 and to analyze those values on the basis of molecular structures of their complexes. (12) Funasaki, N.; Yodo, H.; Hada, S.; Neya, S. Bull. Chem. Soc. Jpn. 1992, 65, 1323, and references therein. (13) Isomaa, B.; Hagerstrand, H.; Paatero, G.; Engblom, A. C. Biochim. Biophys. Acta 1986, 860, 510. (14) Hagerstrand, H.; Isomaa, B. Biochim. Biophys. Acta 1989, 982, 179. (15) Inoue, K.; Sekido, T.; Sano, T. Langmuir 1996, 12, 4644. (16) Dharmawardana, U. R.; Christian, S. D.; Tucker, E. E.; Taylor, R. W.; Scamehorn, J. F. Langmuir 1993, 9, 2258.
10.1021/la9816813 CCC: $19.00 © 2000 American Chemical Society Published on Web 10/21/1999
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Experimental Section Materials. Commercial samples of dodecyltrimethylammonium bromide (DTAB), CH3(CH2)11N+(CH3)3Br-, (Tokyo Kasei Organic Chemicals) and 3-(dimethyldodecylammonio)-1-propanesulfonate (DDAPS), CH3(CH2)11N+(CH3)2(CH2)3SO3-, (Sigma) were used without further purification because they did not give any minimum in the surface tension versus concentration plot. All of R-, β-, and γ-CyDs were purchased from Nacalai Tesque Co. (Kyoto). Their water contents were determined from their dry weights. All inorganic salts used (NaBr, NaCl, NaH2PO4‚ 2H2O, and Na2HPO4‚12H2O) were of analytical grade from Wako Pure Chemicals (Osaka). The ion-exchanged water was used after double distillation. Methods. All experiments were carried out at 310 K in the presence of 140.5 mmol dm-3 (mM) NaCl and 10 mM phosphate buffer (pH 7.4). The surface tension of an aqueous solution containing CyD and a surfactant was measured by the Wilhelmy method. The equilibrium surface tension was used for further analysis. All measurements of surface tension were carried out in the absence of erythrocytes. Human blood was collected from healthy donors, with 0.47% sodium citrate as an anticoagulant. Erythrocytes, separated by centrifugation at 1000g for 10 min, were washed three times with 10 mM isotonic phosphate buffer (pH 7.4) and then suspended in the buffer solution to give a hematocrit value of 10%.17 To 4 cm3 of the buffer solution containing a surfactant (DTAB or DDAPS) or one of the CyDs was added 0.4 cm3 of the erythrocyte suspension. The mixture was incubated for 30 min at 310 K and then centrifuged at 1000g for 30 min. The percent hemolysis was determined from the absorbance at 543 nm due to hemoglobin in the supernatant; 100% hemolysis is the 543 nm absorbance of an erythrocyte solution hemolyzed completely with distilled water.11,18 For some systems of CyD and a surfactant, the 543 nm absorbance exceeded this value by ca. 5%, probably because of potential interaction between hemoglobin and the CyD or surfactant. For such cases, we corrected this effect by regarding the higher absorbance as 100% hemolysis.
Figure 1. (a) Hemolysis by aqueous DTAB solutions and (b) the surface tension of its solutions in 10 mM phosphate buffer (pH 7.4) containing 140.5 mM NaCl at 310 K. The solid line in (b) is calculated from eq 1.
Results and Discussion Suppression of DTAB-Induced Hemolysis and Surface Tension Elevation by CyD. The percent hemolysis by DTAB increases with increasing DTAB concentration (Figure 1a) and reaches 100% at 1.0 mM. DTAB penetrates the erythrocyte membrane, making it fragile as an exogenous membrane component, and results in hemolysis. The surface tension of an aqueous DTAB solution decreases with increasing concentration (Figure 1b). This decrease is ascribed to the adsorption of DTAB to the airwater interface. We did not carry out experiments of hemolysis and surface tensions at higher DTAB concentrations. At a higher concentration DTAB must form its micelle.19 As Figure 1a shows, DTAB completely hemolyzes at 1.0 mM. A final concentration of 1 mM DTAB and an appropriate concentration of CyD was added to the erythrocyte. Next, the percent hemolysis was determined in the solution of 1.0 mM DTAB and CyD as a function of the CyD concentration. The hemolysis induced by 1.0 mM DTAB is suppressed by the addition of any CyD (Figure 2a). The suppression of hemolytic activity by CyD is in the order R-CyD ≈ β-CyD > γ-CyD. This is a remarkable result, because R-CyD did not suppress the percent hemolysis induced by drugs.11 The surface tension of the 1.0 mM DTAB solution in the absence of CyD is increased by the addition of these CyDs, in the same order of CyD (Figure 2b). These changes in (17) Uekama, K.; Irie, T.; Sunada, M.; Otagiri, M.; Iwasaki, K.; Okano, Y.; Miyata, T.; Kase, Y. J. Pharm. Pharmacol. 1981, 33, 707. (18) Fujii, T.; Sato, T.; Tamura, A.; Wakatsuki, M.; Kanaho, Y. Biochem. Pharmacol. 1979, 28, 613. (19) Funasaki, N.; Hada, S.; Neya, S. J. Phys. Chem. 1986, 90, 5469.
Figure 2. Effects of R-CyD (O), β-CyD (0), and γ-CyD (4) on (a) the hemolysis and (b) the surface tension of a 1.0 mM DTAB solution in 10 mM phosphate buffer (pH 7.4) containing 140.5 mM NaCl at 310 K. The surface tension was measured in the absence of CyD. The solid lines in (a) are calculated from eq 11 with the observed surface tension data (b). The solid lines in (b) are calculated from eqs 1 and 9 with binding constant data (Table 1).
hemolysis and surface tension will be mainly due to the reduction of free DTAB concentration induced by the binding of DTAB with CyD. Suppression of DDAPS-Induced Hemolysis and Surface Tension Elevation by CyD. To confirm the above results for DTAB, we carried out the same experiments for DDAPS. As the DDAPS concentration was
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Figure 4. Schematic interrelation among the equilibria of surface adsorption, micellization, CyD inclusion, binding, and lipid extraction for the surfactant-CyD system.
In the absence of CyD and erythrocytes, the surface tension, γ, of an aqueous surfactant solution will be a unique function of the molarity, [S], of the surfactant in the monomer state: Figure 3. Effects of R-CyD (O), β-CyD (0), and γ-CyD (4) on (a) the hemolysis and (b) the surface tension of a 1.6 mM DDAPS solution in 10 mM phosphate buffer (pH 7.4) containing 140.5 mM NaCl at 310 K. The surface tension was measured in the absence of CyD. The solid lines in (a) are calculated from eq 11 with the observed surface tension data (b). The solid lines in (b) are calculated from eqs 1 and 9 with binding constant data (Table 1).
increased, it caused the initiation of hemolysis at 0. 8 mM and complete hemolysis above 1.6 mM (data not shown). The surface tension of an aqueous DDAPS solution decreased with increasing concentration until 3 mM (data not shown). The critical micelle concentration (cmc) will be much higher than this highest concentration.20 Furthermore, we investigated the effects of CyDs on hemolysis by a 1.6 mM DDAPS solution (Figure 3a). The suppression of DDAPS-induced hemolysis by CyDs is in the order R-CyD ≈ β-CyD > γ-CyD. Τhis is the same order as with DTAB (Figure 2a). Figure 3b shows the effects of CyDs on the surface tension of a 1.6 mM DDAPS solution. The surface tension increases gradually at low CyD concentrations and rather abruptly at higher concentrations. The power of surface tension elevation by CyDs at 1.6 mM DDAPS is in the order β-CyD ≈ R-CyD > γ-CyD. Estimation of Binding Constants from Surface Tension Data. We have developed a surface tension method for determining the binding constant for the amphiphile-CyD system,12 and applied it to a surfactant12 and drugs.10,11 Figure 4 shows a schematic model for several equilibria in the surfactant-CyD system. We do not need to consider the micellization of the surfactant, because all experiments were carried out below the cmc. Many surfactants stepwise form 1:1, 1:2, and 2:1 complexes with CyD.12,21-26 (20) Fendler, E. J.; Day, C. L.; Fendler, J. H. J. Phys. Chem. 1972, 76, 1460. (21) Park, J. W.; Song, H. J. J. Phys. Chem. 1989, 93, 6454. (22) Wan Yunus, W. M. Z.; Taylor, J.; Bloor, D. M.; Hall, D. G.; WynJones, E.; J. Phys. Chem. 1992, 96, 8979. (23) Tominaga, T.; Hachisu, D.; Kamado, M. J. F. Langmuir 1994, 10, 4676. (24) Mwakibete, H.; Cristantino, R.; Bloor, D. M.; Wyn-Jones, E.; Holzwarth, J. F. Langmuir 1995, 11, 57.
γ ) f{[S]}
(1)
Here f is an empirical function containing several adjustable parameters. We assumed different functions for DTAB and DDAPS. In the absence of any aggregate, the monomer concentration is equal to the total surfactant concentration CS. The adjustable parameters in function f were determined to be best fit to the observed surface tension data shown in Figure 1b and the corresponding data for DDAPS by nonlinear least-squares method. The best fitting was obtained by minimizing the SS value: N
SS )
∑(γobsd - γcalcd)2
(2)
Here N denotes the number of data (N ) 10 for DTAB and N ) 12 for DDAPS). The solid lines shown in Figure 1b are theoretically calculated from eq 1 with these bestfit parameters. The molarity of the 1:1 complex, SD, is written using the binding constant, K1, of 1:1 complexation as4,12
[SD] ) K1[S][D]
(3)
where [D] denotes the molarity of free CyD. The molarities of 1:2 and 2:1 complexes are also written as
[SD2] ) K1K2[S][D]2
(4)
[S2D] ) K1K3[S]2[D]
(5)
The total concentration of surfactant is expressed as
CS ) [S] + K1[S][D] + K1K2[S][D]2 + 2 K1K3[S]2[D] (6) Here the adsorption amount of surfactant at the airwater interface is neglected. The total concentration of (25) Shen, X.; Βelletete, M.; Durocher, G. Langmuir 1997, 13, 5830. (26) Wilson, L. D.; Verrall, R. E. Can. J. Chem. 1998, 76, 25.
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Table 1. Binding Constants of Surfactants and Drugs with r-, β-, and γ-CyD K1 (M-1)
K2 (M-1)
K3 (M-1)
γ-
17 000 17 000 17 000 18 100 23 700 2400 110
1000 -
1820
Rβγ-
19 000 26 000 240
-
2000
Rβγ-
43 000 51 200 570
3100 -
5570
Rβγ-
19 000 26 000 240
1150 -
1380 3080
Rβ-
21 000 33 000
1200 430
-
Rβ-
43 000 26 800 25 600
3100 440 200
-
Rβγ-
110 6200 420
-
2300
Rβγ-
80 3400 230
1200 -
70
CyD Rβ-
a
Phosphate buffer. b Surface tension. c This work.
d
conditions DTAB 141 mM NaCl + 10 mM PB,a 310 K H2O, 298 K 141 mM NaCl + 10 mM PB, 310 K H2O, 298 K H2O, 298 K H2O, 298 K 141 mM NaCl + 10 mM PB, 310 K DDAPS 141 mM NaCl + 10 mM PB, 310 K 141 mM NaCl + 10 mM PB, 310 K 141 mM NaCl + 10 mM PB, 310 K tetradecyltrimethylammonium bromide H2O, 298 K H2O, 298 K H2O, 298 K dodecyl maltoside H2O, 293 K H2O, 293 K H2O, 293 K sodium dodecanoate D2O, 295 K D2O, 295 K sodium dodecyl sulfate H2O, 298 K H2O, 298 K H2O, 298 K chlorpromazine hydrochloride 141 mM NaCl + 10 mM PB, 310 K 141 mM NaCl + 10 mM PB, 310 K 141 mM NaCl + 10 mM PB, 310 K propantheline bromide 141 mM NaCl + 10 mM PB, 310 K 141 mM NaCl + 10 mM PB, 310 K 141 mM NaCl + 10 mM PB, 310 K
ref
STb EMFd ST EMF ITCe ECf ST
c 22 c 22 24 27 c
ST ST ST
c cc c
EMF EMF EMF
23 23 23
ST ST ST
12 12 12
NMR NMR
26 26
FLg FL FL
25 25 21
ST ST ST
11 11 11
ST ST ST
10 10 10
Electromotive force. e Isothermal calorimetry. f Electric conductance. g Fluorescence.
CyD is written as
CD ) [D] + K1[S][D] + 2 K1K2[S][D]2 + K1K3[S]2[D] (7) The concentration [D] of free CyD can be obtained from
[D] ) {-1 -K1[S] - K1K3[S]2 + [(1 + K1[S] + K1K3[S]2)2 +8CDK1K2[S])1/2}/4 K1K2[S] (8) Substitution of eq 8 into eq 6 yields:
CS ) g{[S]}
method
(9)
Because g is a rather complicated function containing CD, K1, K2, and K3, we do not write it in explicit form. More complicated cases for multiple binding and self-associating equilibria have been reported elsewhere.9-12 CyD is surface-inactive enough not to change the surface tension of water. Therefore, we presume that all the complexes of surfactant and CyD are also surfaceinactive.10-12 Then eq 1 will hold true even in the presence of CyD. The observed surface tension data for DTAB shown in Figure 2b were analyzed, taking into consideration adequate complex species. The theoretical surface tension for a DTAB solution at a given set of CS ) 1.0 mM and CD was obtained by regarding a set of values of K1, K2, and K3 as adjustable parameters. The binding constants thus obtained are shown in Table 1. For R- and β-CyDs, the binding models taking into consideration 1:2 and 2:1
complex species did not improve fitting significantly. The fitting procedure has already been reported in detail.10,12 The same analysis was carried out for the DDAPS-CyD systems, and their binding constants are also included in Table 1. Relation between Binding Constants and Molecular Structures. Table 1 summarizes our and relevant literature binding constants for several surfactants12,21-27 and two drugs.10,11 The electric conductance method generally gave very small binding constants (e.g., see the DTAB-β-CyD system in Table 1).12,21,27 Most of the binding constants have been determined at 298 K. As is evident from Table 1, our data at 310 K are smaller than the literature values at 298 K. This is a general tendency, indicating that CyD inclusion is an exogenous reaction: the enthalpy of complexation is negative. For most of the surfactants having a long alkyl chain, the magnitude of the 1:1 binding constant for CyDs is in the order β-CyD g R-CyD . γ-CyD.12,21,23 Reliable 1:2 and 2:1 binding constant data are still much less than 1:1 data, especially for γ-CyD. We have proposed probable structures of several surfactant-CyD complexes.12 Little is known about the quantitative relation between multiple binding constants and structures of complexes. However, we can find some general tendencies in Table 1. For surfactants, the magnitude of the 1:2 binding constant, K2, for CyDs is in the order R-CyD > β-CyD > γ-CyD. The 1:2 binding constant is smaller than the 1:1 constant by one order or more, suggesting that the second complexation of CyD is inhibited by steric hindrance. For the surfactant-γ-CyD system, the 2:1 binding (27) Junguera, E.; Pena, L. Aicart, E. Langmuir 1995, 11, 4685.
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constant K3 is much larger than K1: the second surfactant molecule cooperatively binds to the 1:1 complex of surfactant and γ-CyD. Although the cavity of γ-CyD is too wide for a single alkyl chain, it is best accommodated for two alkyl chains. The 2:1 complexation can be regarded as the dimerization of a surfactant in the CyD cavity. Surfactants form dimers, though their dimerization constants are small.28-30 The dimeric structures of the surfactant in water and the CyD cavity would be similar to each other.12 It is likely that compounds with larger dimerization constants have larger K3 values, though there are few systematic studies.11 From Table 1 we can suggest that the binding constants for 1:2 and 2:1 complexation decrease with increasing temperature. Chlorpromazine hydrochloride (CPZ) is more hydrophobic than propantheline bromide (PB).11 This difference is also reflected in the binding behavior. As Table 1 shows, the binding constants of CPZ with R-, β-, and γ-CyDs are larger than those of PB. CPZ binds to γ-CyD cooperatively (K3 > K1), whereas PB binds anti-cooperatively (K3 < K1). The larger dimerization constant of CPZ,11,31,32 compared to PB,10,33,34 is one of the driving forces for the cooperative binding of CPZ. The dimer of CPZ is formed by stacking interactions of its phenothiazine ring in water.31 Both the homodimers of CPZ and PB in the γ-CyD cavity will have three-dimensional structures in which their tricyclic rings stack in parallel arrangements.10,11 Thus, CPZ can form the 2:1 complex with γ-CyD, without significant changes in the three-dimensional structure of dimer. This favors the cooperative binding of CPZ to γ-CyD. However, two xanthene rings of the PB dimer in water are in perpendicular arrangements, though those in the 2:1 complex are in parallel arrangements.34 Though the present study on DTAB and DDAPS provides useful data to understand the interaction between surfactants and CyDs, the 1:1, 1:2, and 2:2 binding constants for those systems merit further investigations.12 Prediction of Hemolysis from Surface Tension Data. CyDs can hemolyze at much high concentrations: 50% hemolysis is 11 mM for R-CyD, 5 mM for β-CyD, and 29 mM for γ-CyD.6,11 No CyD induces hemolysis at the low concentrations investigated in this work. If CyD and its complexes with a surfactant have no influence for erythrocytes, the percent hemolysis by solutions containing surfactant, CyD, and erythrocytes will be determined only by the concentration of free surfactant:
percent hemolysis ) h([S])
(10)
The function h can be determined from the observed hemolysis data of each surfactant (Figure 1a for DTAB and the corresponding result for DDAPS), if the adsorption amount of surfactant to erythrocytes is neglected. This assumption is justified approximately, if all hemolysis experiments are carried out at a low concentration of (28) Funasaki, N. Adv. Colloid Interface Sci. 1993, 43, 87. (29) Funasaki, N.; Hada, S.; Neya, S. Trends Phys. Chem. 1997, 6, 253. (30) Funasaki, N.; Shim, H.-S.; Hada, S. J. Phys. Chem. 1992, 96, 1998. (31) Attwood, D.; Waigh, R.; Blundell, R.; Bloor, D.; The´vand, A.; Boitard, E. Dube`s, J.-B.; Tachoire, H. Magn. Reson. Chem. 1994, 32, 468. (32) Funasaki, N.; Hada, S.; Paiement, J. J. Phys. Chem. 1991, 95, 4131. (33) Funasaki, N.; Uemura, Y.; Hada, S.; Neya, S. Langmuir 1996, 12, 2214. (34) Hada, S.; Ishikawa, S.; Neya, S.; Funasaki, N. J. Phys. Chem. B 1999, 103, 2579.
Figure 5. Relation between the hemolysis and surface tension in the absence (b) and presence of R-CyD (O), β-CyD (0), and γ-CyD (4) for DTAB and DDAPS. All data of surface tensions and percent hemolysis determined in the present work (the results shown in Figures 1-3 and for DDAPS alone) are included.
erythrocytes. Then a combination of eqs 1 and 10 yields
percent hemolysis ) h[f--1(γ)]
(11)
According to this equation, the percent hemolysis by solutions containing a surfactant and CyD is a unique function of surface tension, regardless of the surfactant concentration and the kind and concentration of CyD. To confirm eq 11, we plotted all surface tension data shown in Figures 1b, 2b, and 3b against the hemolysis data shown in Figures 1a, 2a, and 3a. As Figure 5 shows, the hemolysis data fall on a master line for DTAB and DDAPS each. Here it is noted that the functions f and h depend on each surfactant and drug.11 On the basis of this result, we can calculate the percent hemolysis of the solution from the observed surface tension value of a mixed DTAB and CyD solution (Figure 2b) by using eqs 1 (based on the data in Figure 1b) and 11. Namely, we can determine the relation between the percent hemolysis and the surface tension of the DTAB solution alone (Figure 1). Then, we predict the percent hemolysis of a mixed DTAB and CyD solution from this relation and the observed surface tension of the solution. As the solid line in Figure 2a shows, it is in very good agreement with the observed hemolysis percent for all the CyDs. The solid line in Figure 3a for DDAPS shows the prediction based on eqs 1 (data not shown) and 11. This prediction also holds true for all the CyDs used, although it failed for R-CyD in the cases of CPZ and PB. This exception for R-CyD was interpreted in terms of stronger competitive binding of membrane phospholipid than these drugs.11 This interpretation is verified, because DTAB and DDAPS have high affinities for all the CyDs (Table 1). Implications and Limitations of the Present Approach. Chemistry is rapidly extending toward the field of life sciences. There we need more physicochemical data at 310 K, body temperature, instead of 298 K. Binding constants for most CyD inclusion systems have been determined at 298 K. To analyze various functions of CyDs in human bodies quantitatively, we need binding constant data at 310 K. If we used surface tension data at 298 K, those data would not quantitatively correlate with the percent hemolysis, such as shown in Figures 2a and 3a. Surface tension can be used to determine binding constants and to predict hemolysis suppression by CyD. This method is applicable to self-associable guests and
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any multiple-complexing systems, because it is based on the assumption that the surface tension and the percent hemolysis depend on the free surfactant concentration alone.11,12 For example, this method is applicable to the DDAB-γ-CyD system forming the 2:1 complex and to the PB system forming the self-association.10 If the free guest concentration in a CyD solution is estimated from binding constants obtained by any method, it can be used to predict the percent hemolysis in the mixed solution. However, it is not generally easy to determine such binding constants and self-association constants. Our method is independent of the details of stoichiometry and self-association, though it would be inapplicable to surface-active CyDs. The surface tension method for predicting the hemolysis suppression by CyD is based on eq 10, which neglects the decrease in surfactant concentration due to the adsorption to the aqueous surface and the erythrocyte, the hemolysis by CyDs, and the binding of membrane phospholipids. The decrease due to the aqueous surface adsorption is negligible except for extremely surface-active substances.35 The decrease due to the erythrocyte adsorption can be reduced in dilute erythrocyte solutions. The present
Funasaki et al.
prediction, however, is inapplicable both to systems containing so high CyD concentrations as to induce hemolysis (the PB-γ-CyD system) and to the system having a small binding constant (the PB-R-CyD system).11 The present approach will generally apply to other surfactants and surface-active drugs. When we use a surfactant for the formulation of pharmaceuticals and foods, it will be very hemolytic. If we could reduce its toxicity by the addition of CyD, it might be utilized commercially. Then, it is desirable that we be able to determine the kind and concentration of CyD on the basis of some physicochemical measurements and fewer hemolysis experiments. Acknowledgment. Thanks are due to Ms. Kaori Oneda for her valuable help with some experiments of DDAPS. LA9816813 (35) Funasaki, N.; Hada, S.; Suzuki, K. Chem. Pharm. Bull. 1976, 24, 731.
