Langmuir 1997, 13, 6115-6119
6115
Exclusion of the Local Anesthetic Procaine Hydrochloride from a Surface-Adsorbed Film and Micelle of Decylammonium Chloride Hitoshi Matsuki,* Kensaku Shimada, and Shoji Kaneshina Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Minamijosanjima, Tokushima 770, Japan
Michio Yamanaka Department of Chemistry, Faculty of Science, Kyushu University Ropponmatsu, Ropponmatsu, Fukuoka 810, Japan
Hiroshi Kamaya and Issaku Ueda Department of Anesthesia, DVA Medical Center, and University of Utah School of Medicine, Salt Lake City, Utah 84148 Received June 17, 1997. In Final Form: September 4, 1997X Incorporation of the local anesthetic procaine hydrochloride (PC‚HCl) into surface-adsorbed films and micelles formed by decylammonium chloride (DeAC) was investigated by measuring the surface tensions of aqueous solutions of a DeAC-PC‚HCl mixture. The thermodynamic quantities of the miscibility in these molecular aggregates, the total surface density, and the compositions of PC‚HCl in the surfaceadsorbed film and micelle were calculated by applying thermodynamic equations to the surface tension data. The phase diagrams of surface adsorption and micelle formation showed a peculiar feature; namely, the compositions of PC‚HCl in the adsorbed film and micelle showed negative values over the whole composition range measured. The negative values were caused by the exclusion of the PC‚HCl molecules from the adsorbed film and the micelle of DeAC. PC‚HCl was not incorporated into such hydrophobic environments. In contrast, tetracaine hydrochloride (TC‚HCl) was incorporated into the adsorbed film and micelle of DeAC at all DeAC-TC‚HCl compositions. The difference in incorporation between TC‚HCl and PC‚HCl is attributable to the hydrophobicity of their molecules.
Introduction Local anesthetics must be soluble in water and can be incorporated into biological membranes.1 Many reported the incorporation of local anesthetics into multilamellar vesicles of phospholipid bilayers. Molecular aggregates formed by surfactants produce microscopic hydrophobic environments similar to phospholipid bilayers, and acquisition of the experimental values of the aggregates at a thermodynamic equilibrium is easier than that for bilayers. In our previous paper,2 we examined incorporation of micelle-forming local anesthetics, dibucaine hydrochloride (DC‚HCl) and tetracaine hydrochloride (TC‚HCl), into surface-adsorbed films and micelles of molecular aggregates formed by cationic surfactant as model biomembranes. It was shown that the anesthetics having stronger hydrophobicity are incorporated into hydrophobic environments of the molecular aggregates. Incorporation into the surface-adsorbed film was less than that into the micelle due to the larger polar head group of the anesthetic molecules, which favors interaction with globular structure. Although all local anesthetics are hydrophobic, the degree of hydrophobicity varies among agents. Since the anesthetic potency of local anesthetics correlates with their hydrophobicity, it is important to study the incorporation of various local anesthetics with different hydrophobicities. * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, October 15, 1997. (1) Attwood, D.; Florence, A. T. Surfactant Systems; Chapman and Hall: London, 1983; Chapter 4, pp 153-155. (2) Matsuki, H.; Hashimoto, S.; Kaneshina, S.; Yamanaka, M. Langmuir 1997, 13, 2687.
S0743-7463(97)00643-4 CCC: $14.00
We compared the incorporation into the model membranes of a local anesthetic having a weaker hydrophobicity, procaine hydrochloride (PC‚HCl), with that of a local anesthetic having a stronger hydrophobicity, TC‚HCl. Reports on the effect of anesthetics having weak hydrophobicities on model membranes are few compared to those about anesthetics with strong hydrophobicities.3-15 Few studies have been reported about the surface tension of procaine on the model membrane. Lin and Ueda et al.16 examined the binding of procaine on the phospholipid monolayer membranes and analyzed the experimental data by statistical thermodynamics. Tomoaia-Cotisel and Cadenhead17 studied the interaction of procaine with (3) Papahadjopoulos, D.; Jacobson, K.; Poste, G.; Shepherd, G. Biochim. Biophys. Acta 1975, 394, 504. (4) Fernandez, M. S. Biochim. Biophys. Acta 1980, 597, 83. (5) Fernandez, M. S. Biochim. Biophys. Acta 1981, 646, 27. (6) Eftink, M. R.; Puri, R. K.; Ghahramani, M. D. Biochim. Biophys. Acta 1985, 813, 137. (7) Frezzatti, W. A., Jr.; Toselli, W. R.; Schreier, S. Biochim. Biophys. Acta 1986, 860, 531. (8) Auger, M.; Jarrell, H. C.; Smith, I. C.; Wong, P. T.; Siminovitch, D. J.; Mantsch, H. H. Biochemistry 1987, 26, 8513. (9) Auger, M.; Jarrell, H. C.; Smith, I. C. Biochemistry 1988, 27, 4660. (10) Auger, M.; Jarrell, H. C.; Smith, I. C.; Siminovitch, D. J.; Mantsch, H. H.; Wong, P. T. Biochemistry 1988, 27, 6086. (11) Seelig, A.; Allegrini, P. R.; Seelig, J. Biochim. Biophys. Acta 1988, 939, 267. (12) Bottner, M.; Winter, R. Biophys. J. 1993, 65, 2041. (13) Louro, S. R. W.; Nascimento, O. R.; Tabak, M. Biochim. Biophys. Acta 1994, 1190, 319. (14) Peng, X.; Jonas, J. Biochemistry 1992, 31, 6383. (15) Peng, X.; Jonas, A.; Jonas, J. Chem. Phys. Lipids 1995, 75, 59. (16) Lin, H.- C.; Ueda, I.; Lin, S. H.; Shieh, D. D.; Kamaya, H.; Eyring, H. Biochim. Biophys. Acta 1980, 598, 51. (17) Tomoaia-Cotisel, M.; Cadenhead, D. A. Langmuir 1991, 7, 964.
© 1997 American Chemical Society
6116 Langmuir, Vol. 13, No. 23, 1997
Matsuki et al.
stearic acid monolayers at the air/water interface. Recently, Makino et al.18 reported the effect of procaine on the dynamic behavior of a phospholipid thin film by measuring the dynamic surface pressure and surface area characteristics. In the present study, the incorporation of PC‚HCl into a surface-adsorbed film and micelle of cationic surfactant, decylammonium chloride (DeAC), is investigated by measuring the surface tension of the aqueous solutions of the DeAC-PC‚HCl mixture. The compositions of PC‚HCl in the molecular aggregates are calculated by applying the thermodynamic equations to the surface tension data. We discuss the incorporation of PC‚HCl into the molecular aggregates by the phase diagrams of surface adsorption and micelle formation, which are obtained from the calculated composition values. The incorporation of PC‚HCl into the aggregates is compared with that of TC‚HCl, which is similar in molecular structure to PC‚HCl but has a stronger hydrophobicity.
Figure 1. Surface tension vs total molality curves at constant composition: (X2) (1) 0; (2) 0.300; (3) 0.500; (4) 0.670; (5) 0.800; (6) 0.880; (7) 0.910; (8) 0.950; (9) 1.
Experimental Section Materials. Procaine (2-(diethylamino)ethyl p-(amino)benzoate) hydrochloride (PC‚HCl) was purchased from Sigma Chemical Company and purified by repeated recrystallizations from ethanol. Decylammonium chloride (DeAC) was synthesized and purified by the method reported previously.2 Their purities were confirmed by elemental analysis and by observing the absence of a minimum on the surface tension vs concentration curves near the critical micelle concentration. Water was distilled three times after deionization, where the second and third stages were from dilute alkaline permanganate solution. Automatic Surface Tension Measurements. An automatic surface tension measurement apparatus based on the drop volume method19 was used to measure the surface tensions of aqueous surfactant-anesthetic solutions. The densities of the aqueous solutions were measured by a vibrating-tube density meter (Anton Paar DMA60/602) described previously.20 The experimental error for the value of surface tension was less than 0.05 mN m-1. All the measurements were performed at 298.15 ( 0.01 K under atmospheric pressure. The surface tension γ of the aqueous solutions was measured as a function of the total molality of DeAC and PC‚HCl, m (mol kg-1 of solvent), and the mole fraction of PC‚HCl in the total components, X2, defined by
m ) m1 + m2
(1)
X2 ) m2/m
(2)
Figure 2. Surface tension vs composition curves at constant total molality (m/mmol kg-1): (1) 40; (2) 50; (3) 60; (4) 80; (5) 100; (6) 150; (7) 250; (8) γC vs X2.
and
where m1 and m2 denote the molalities of DeAC and PC‚HCl, respectively.
Results and Discussion Figure 1 shows the γ vs m curves at constant X2. The γ values decreased rapidly with increasing m. The break point, which corresponds to the critical micelle concentration (cmc), was observed on the γ vs m curve in the composition range from pure DeAC (X2 ) 0) to the mixture of X2 ) 0.910. On the other hand, the γ value of pure PC‚HCl slightly decreased linearly with increasing m. A remarkable difference between the γ vs m curve of PC‚HCl (curve 9) and that of X2 ) 0.950 (curve 8) is seen. The composition dependences of γ at constant m and of m at constant γ are shown in Figures 2 and 3, respectively. Both figures are constructed by estimating the γ values at a given m and the m values at a given γ from the γ vs m curves in Figure 1. The γ values at constant m and the (18) Makino, M.; Kamiya, M.; Nakajo, N.; Yoshikawa, K. Langmuir 1996, 12, 4211. (19) Matsuki, H.; Kaneshina, S.; Yamashita, Y.; Motomura, K. Langmuir 1994, 10, 4394. (20) Matsuki, H.; Hashimoto, S.; Kaneshina, S.; Yamanaka, M. Langmuir 1994, 10, 1882.
Figure 3. Total molality vs composition curves at constant surface tension (γ/mN m-1): (1) 65; (2) 60; (3) 55; (4) 50; (5) 45; (6) 40; (7) 35; (8) C vs X2.
m values at constant γ increased greatly in the higher X2 region, and the slopes of both curves became steeper at larger m and in the lower γ region. Figures 2 and 3 also include the total molality of the cmc (C) vs X2 curve and the surface tension at the cmc (γC) vs X2 curve, which
Exclusion of Local Anesthetic from Surfactant Aggregates
Langmuir, Vol. 13, No. 23, 1997 6117 H where ΓH 1 and Γ2 are the surface densities of DeAC and PC‚HCl. They are defined with reference to the two dividing planes making the excess numbers of moles of water and air.25 As reported previously,24 the ΓH values were estimated from the slope of the γ vs m curves shown in Figure 1 and by the equation
ΓH ) -(m/2RT)(∂γ/∂m)T,p,X2
Figure 4. Total surface density vs total molality curves at constant composition (X2): (1) 0; (2) 0.300; (3) 0.500; (4) 0.670; (5) 0.800; (6) 0.880; (7) 0.910; (8) 0.950; (9) 1; (b) total surface density at the cmc (ΓH,C).
were determined from the break point on the γ vs m curve in Figure 1. A rapid increase of the C value was observed with increasing X2, whereas the γC value decreased slightly with an increase in X2. Both DeAC and PC‚HCl are salts of a tertiary amine and hydrogen chloride; hence, decylammonium and procaine cations are actually weak electrolytes and dissociate partially:
AH+ S A + H+
(3)
where AH+ and A are the protonated (charged) and free base (uncharged) forms, respectively. The ratio of charged and uncharged molecules in the aqueous solution is determined by the Henderson-Hasselbalch equation
pH ) pKa + log[A]/[AH+]
(4)
We assumed that DeAC and PC‚HCl are uni-univalent electrolytes because they have relatively high pKa values (DeAC (11.64),21 PC‚HCl (9.05)22 ) and the pH values of the aqueous solutions were between 5.0 and 5.5. The effect of partial dissociation of both cations is negligible from eq 4. The quantities of PC‚HCl incorporated into surfaceadsorbed films and micelles of DeAC can be revealed by investigating the miscibility of DeAC and PC‚HCl in the aggregates.23,24 The miscibilities at concentrations below the cmc and at the cmc are described in the following sections. Incorporation of PC‚HCl into the Surface-Adsorbed film of DeAC. Incorporation of PC‚HCl into surface-adsorbed films of DeAC at concentrations below the cmc is elucidated by two surface quantities: total surface density, ΓH, of DeAC and PC‚HCl and the mole fraction of PC‚HCl in the surface-adsorbed film, XH 2. Here ΓH and XH are defined respectively by 2 H ΓH ) ΓH 1 + Γ2
(5)
H H XH 2 ) Γ2 /Γ
(6)
and
(21) Hoerr, C. W.; McCorkle, M. R.; Ralston, A. W. J. Am. Chem. Soc. 1943, 65, 328. (22) Kamaya, H.; Hayes, J. J., Jr.; Ueda, I. Anesth. Analg. 1983, 62, 1025. (23) Motomura, K.; Ando, N.; Matsuki, H.; Aratono, M. J. Colloid Interface Sci. 1990, 139, 188. (24) Motomura, K.; Aratono, M. In Mixed Surfactant Systems; Ogino, K., Abe, M., Eds.; Marcel Dekker: New York, 1993; p 99.
(7)
The variation in ΓH with m at constant X2 is shown in Figure 4. We notice that the ΓH values increase up to the values of saturated adsorption with increasing m and shift to the right with increasing X2. This behavior is in contrast to the slight increase of the ΓH value observed for pure PC‚HCl. Another surface quantity, XH 2 , can be evaluated directly from the m vs X2 curve at constant γ given in Figure 3 by the following equation24
XH 2 ) X2 - (2X1X2/m)(∂m/∂X2)T,p,γ
(8)
The resulting phase diagrams of surface adsorption, which are obtained from the m vs XH 2 curves (broken line) and the m vs X2 curves (continuous line), are shown in Figure 5. In the figure are shown the corresponding previous results of the DeAC-TC‚HCl system.2 Interestingly, all H XH 2 values showed negative signs and the absolute X2 values increased with increasing X2 (increasing m). In addition, the shape of the phase diagrams for the DeACPC‚HCl system was remarkably different from the cigar shape diagrams for the DeAC-TC‚HCl system. The definition of XH 2 given in eq 6 indicates that this finding H is closely related to the sign of ΓH 1 and Γ2 of both systems. To clarify the difference between PC‚HCl and TC‚HCl in incorporation into the surface-adsorbed film of DeAC, H we compared the surface densities ΓH 1 and Γ2 of both systems. The dependence of ΓH on X2 at constant γ is obtained from the m vs X2 curve in Figure 3 and the ΓH H vs m curve in Figure 4. The values of ΓH 1 and Γ2 can be H estimated by combining this Γ vs X2 curve with the m vs composition curves in Figure 5. The estimated ΓH, ΓH 1, -1 and ΓH values for both systems at γ ) 50 mN m are 2 plotted against X2 in Figure 6. It is noteworthy that the H behaviors of the ΓH 1 vs X2 and the Γ2 vs X2 curves for both systems are quite different from each other though the ΓH values for both systems decrease with increasing X2. The ΓH 2 value of PC‚HCl is negative over the whole composition range measured at γ ) 50 mN m-1 while the ΓH 2 of TC‚HCl is positive. The peculiar behavior of the m vs XH 2 curves of the DeAC-PC‚HCl system is attributable to the negative adsorption of PC‚HCl. The PC‚HCl molecules adsorb positively at the water/air interface in the absence of DeAC molecules from the ΓH values given in Figure 4, whereas they adsorb negatively in the presence of DeAC, as seen in Figure 6. This suggests that the PC‚HCl molecules are excluded from the surface-adsorbed film formed by DeAC molecules and are not incorporated into the adsorbed film of DeAC at all. It is noted that the remarkable difference between curves 8 and 9 in Figure 1 is caused by the difference in adsorption of PC‚HCl at the water/air interface. On the other hand, the ΓH 2 value of TC‚HCl in the DeAC-TC‚HCl system became positive over the whole composition range. At all compositions the TC‚HCl molecules are incorporated into the surfaceadsorbed film of DeAC. The contrasting results of the two systems are attributable to the difference in hydro(25) Motomura, K. J. Colloid Interface Sci. 1978, 64, 348.
6118 Langmuir, Vol. 13, No. 23, 1997
Matsuki et al.
Figure 5. Total molality vs composition curves at constant surface tension: (A) DeAC-PC‚HCl system; (B) DeAC-TC‚HCl system. γ/mN m-1: (1) 60; (2) 55; (3) 50; (4) 45. (s) m vs X2; (- - -) m vs XH 2.
phobicity between PC‚HCl and TC‚HCl molecules.20 TC‚HCl has a larger hydrophobic group on the aromatic ring than that of PC‚HCl, so that TC‚HCl can be incorporated into the adsorbed film of DeAC more easily than PC‚HCl. Exclusion of PC‚HCl from the adsorbed film of DeAC seems to be caused by the strong hydrophilicity of the amino group of the aromatic ring in the molecule in addition to that of the ionic polar head group. Incorporation of PC‚HCl into the Micelle of DeAC. Since PC‚HCl is not incorporated into the surface-adsorbed film of DeAC, we next studied whether the PC‚HCl molecules are incorporated into micelles formed by DeAC at the cmc. The total aggregation number of DeAC and PC‚HCl, NM, and the mole fraction of PC‚HCl in the micelle, XM 2 , are defined as with the surface-adsorbed film by23,24 M NM ) NM 1 + N2
and
(9)
Figure 6. Surface density vs composition curves at γ ) 50 mN m-1: (A) DeAC-PC‚HCl system; (B) DeAC-TC‚HCl system. H (1) ΓH; (2) ΓH 1 ; (3) Γ2 . M M XM 2 ) N2 /N
(10)
M respectively. Here NM 1 and N2 are the aggregation numbers of DeAC and PC‚HCl in one micelle particle, which are defined with reference to the spherical dividing surface chosen so as to make that of water zero.26 The M respective NM 1 and N2 values cannot be evaluated, but M the X2 values can be obtained by the following relation24
XM 2 ) X2 - (2X1X2/C)(∂C/∂X2)T,p
(11)
The numerical values of XM 2 are calculated by application of eq 11 to the C vs X2 curve in Figure 3. The phase diagram of micelle formation, which is drawn in terms of the values of X2 and XM 2 , is illustrated together with that of the corresponding DeAC-TC‚HCl system in Figure 7. The phase diagrams of micelle formation for both systems were similar in shape to those of surface adsorption: the XM 2 value is negative for the DeAC-PC‚HCl system while it is positive for the DeAC-TC‚HCl system. It is evident (26) Motomura, K.; Yamanaka, M.; Aratono, M. Colloid Polym. Sci. 1984, 262, 948.
Exclusion of Local Anesthetic from Surfactant Aggregates
Langmuir, Vol. 13, No. 23, 1997 6119
Figure 8. Surface tension at the cmc vs composition curves: H,C C (1) γC vs X2; (2) γC vs XM 2 ; (3) γ vs X2 . Figure 7. Critical micelle concentration vs composition curves: (1) DeAC-PC‚HCl system; (2) DeAC-TC‚HCl system. (s) C vs X2, (- - -) C vs XM 2.
that the PC‚HCl molecules are not incorporated into the micelle of DeAC as in the adsorbed film. Yamanaka et al.27 have recently evaluated the miscibility of dodecylammonium chloride (DAC) and sodium chloride (NaCl) in the surface-adsorbed film and micelle. They showed from the phase diagrams of surface adsorption and micelle formation that NaCl is expelled from surface-adsorbed films and micelles formed by DAC. Because the phase diagrams of the DeAC-PC‚HCl system have a striking resemblance to those of the DAC-NaCl system, the influence of PC‚HCl on the miscibility in the adsorbed film and micelle is almost equivalent to that of a uniunivalent inorganic salt. The incorporation of PC‚HCl into the adsorbed film is compared with that into the micelle at the cmc though PC‚HCl is excluded from hydrophobic environments. The mole fraction of PC‚HCl in the surface-adsorbed film at M 24 the cmc, XH,C 2 , is related to the X2 values by the equation H,C XH,C ) XM )(∂γC/∂X2)T,p 2 2 - (X1X2/RTΓ
(12)
where the superscript C represents the thermodynamic quantity at the cmc. The XH,C values are calculated by 2 applying eq 12 to the γC vs X2 curve in Figure 2 and from the ΓH,C and XM 2 values in Figures 4 and 7. Figure 8 shows the γC values plotted against three compositions, X2, H,C H,C XM and XM 2 , and X2 . Both X2 2 have negative values, but the former is larger (smaller negative value) than the latter. The PC‚HCl molecules are expelled from the micelle more than from the adsorbed film. This may arise from differences in the geometrical shape of the interface between the adsorbed film and the micelle, and the result is consistent with that of the DAC-NaCl system reported by Yamanaka et al.27 In the case of the DeAC-TC‚HCl system, the TC‚HCl molecules were incorporated into the micelle more than into the adsorbed film.2 The incorporation of local anesthetics is greatly dependent on the (27) Yamanaka, M.; Matsuki, H.; Ikeda, N.; Aratono, M.; Motomura, K. Langmuir 1994, 10, 2950. (28) Strichartz, G. R.; Sanchez, V.; Arthur, G. R.; Chafetz, R.; Martin, D. Anesth. Analg. 1990, 71, 158. (29) Langerman, L.; Bansinath, M.; Grant, G. J. Anesth. Analg. 1994, 79, 490. (30) Strichartz, G. R. In NEURAL BLOCKADE in Clinical Anesthesia and Management of Pain, 2nd ed.; Cousins, M. J., Bridenbaugh, P. O., Eds.; J. B. Lippincott Company: Philadelphia, PA, 1988; p 32.
hydrophobicity of the anesthetic molecules and the geometry of the aggregates in which the anesthetic molecules are incorporated. Finally we consider the relation between the anesthetic potency of local anesthetics observed in clinical experiments and the incorporation of local anesthetics. Many reports show that the magnitude of anesthetic action is well correlated to the hydrophobicity of the local anesthetic used, and the clinical anesthetic potency of PC‚HCl is known to be much weaker than that of TC‚HCl.28,29 According to the molecular mechanism of local anesthesia,30 local anesthetic molecules must be transferred into the hydrophobic environment of biological membranes from the solution and expand the membranes before local anesthetic action occurs. Present results for the incorporation of PC‚HCl seem to contradict the above mechanism because PC‚HCl has weak clinical anesthetic potency. However, local anesthetic cations partially dissociate into charged and uncharged forms, as given in eq 3, and we only focused our attention on the charged form (procaine cation) in this study. Taking into account that the physiological pH value in vivo has a value of ca. 7.4, it is considered from eq 4 that the uncharged form (procaine free base) in vivo appreciably increases as compared with that in this study and the incorporation is no longer negligible in vivo. The uncharged form will be easily incorporated into the membrane compared with the case for the charged one because of the disappearance of electrostatic interaction with the polar head group. Conclusions Surface tension was measured on the aqueous solution of the DeAC-PC‚HCl mixture to study the incorporation of a local anesthetic having weak hydrophobicity into the model membranes formed by a surfactant. The phase diagrams of the surface adsorption and micelle formation were constructed by the composition values of PC‚HCl in the surface-adsorbed film and the micelle calculated thermodynamically. The diagrams showed that the PC‚HCl molecules were not incorporated into the hydrophobic environments provided by molecular aggregates of DeAC, and they are excluded from such environments. This result contrasts with that of an anesthetic having strong hydrophobicity such as TC‚HCl. Local anesthetics exist in two forms in the solution at physiological pH. The hydrophobic structure and pKa of the molecules determine the local anesthetic potency. Transferring local anesthetic molecules into hydrophobic environments in biological membranes is essential for the local anesthetic action. LA9706431
