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Pre-micelle and micelle formation of local anesthetic dibucaine hydrochloride
Studies in Surface Science and Catalysis 132 Y. Iwasawa, N. Oyamaand H. Kunieda (Editors) 'c:> 2001 Elsevier Science B.V. All rights reserved.
113
Pre-micelle and micelle formation of local anesthetic dibucaine hydrochloride Hitoshi Matsukia, Takahiro Miyata^, Toshiharu Yoshioka^, Hiromu Satake*> and Shoji Kaneshinaa ^Department of Biological Science and Technology, Faculty of Engineering, and ^'Center for Cooperative Research, The University of Tokushima, Minamijosanjima, Tokushima 770-8506, Japan The molecular-aggregate formation of local anesthetic dibucaine hydrochloride (E)C»HC1) was investigated from the electrode potential and light scattering measurements of the aqueous solutions. The electrode potential of E)C»HC1 solutions showed the deviation from Nemstian response at low concentrations below the critical micelle concentration (CMC). We considered the possibility of pre-micelle formation of DC»HC1 in the solution and found that dibucaine cation forms a trimer with two chloride ions in the concentration range. On the other hand, the aggregation number of 15 were obtained for DC^HCl micelles in water from the light scattering measurements. Five dibucaine trimers associate cooperatively and form the micelle at concentrations above the CMC. Further, we observed the E)C»HC1 micelle grow one-dimensional direction like small rod-hke micelle in solutions of high ionic strength. The pre-micelle, micelle formation and micellar growth of dibucaine may be attributable to the stacking of dibucaine molecules due to the ;r electron interaction of an aromatic ring in the molecule. 1. INTRODUCTION Local anesthetics have tertiary amine structures with an aromatic ring. Some with large hydrophobicities form molecular aggregates such as micelles in aqueous solutions. The potency of local anesthetics to block nerve conduction is based on their ability to associate with membranes. There are numerous reports demonstrating that the clinical potency of local anesthetics correlates extremely well with their ability to adsorb to lipid monolayers and bilayers. The formation of molecular aggregates and the partitioning into hydrophobic environments of cell membranes, which is an important process for local anesthesia, are both based on the hydrophobicity. The micelle formation of local anesthetics in aqueous solutions has beenreportedby several researchers [1-4]. They showed that the aggregation of local anesthetics is different from that of surfactants with a straight hydrophobic chain. Recently, we have also shown the existence of micellar aggregates for local anesthetics in the aqueous solution by ionic activity, differential scanning calorimetry (DSC), density, and surface tension measurements [5], etc. In the present study, we examined the formation of pre-micellar and micellar aggregates for dibucaine hydrochlorides (DC»HC1) from the electrode potenrial and light scattering measurements of the aqueous solutions. The aggregation behavior of DC^HCl in the aqueous solution at concentrations below and above the critical micelle concentration (CMC) and that in solutions of high ionic strength was characterized.
114 2. EXPERIMENTAL Dibucaine hydrochloride (2-butoxy-N-[2-(diethylamino)ethyl]-4-quinolinecarboxamide hydrochloride: C2oH29N302»HCl) was purchased from Sigma Chemicals (St. Louis, MO) and purified by the method described previously [6]. Water was distilled twice after deionization. The coated-wire electrode selectively sensitive to dibucaine cation was prepared by the same method described previously [7]. The electromotive force (EMF) of the dibucaine cation and chloride ion (Toko Chemicals' electrodes 7020) was measured by a digital multi-ion monitor (Yamashita Giken Co. Ltd.,Tokushima, Japan) at 298.15 K under atmospheric pressure [7]. Static and dynamic light scattering measurements were performed by light-scattering spectrophotometer DLS-7000 (Otsuka Electronics, Osaka, Japan) with Ar and He-Ne lasers. The scattered light of aqueous dibucaine solutions in the absence and presence of sodium chloride was measured under the same condition as the EMF measurements. The increment of refractive index for dibucaine solutions was measured by differential refractometer DRM-1021 (Otsuka Electronics) to calculate the weight average of molecular weight for dibucaine. 3. RESULTS AND DISCUSSION 3.1. Pre-micelle formation of DC-HCI at concentrations below the CMC The electrode responses of dibucaine cation (DC»H+) and chloride ion (CI) in the aqueous solution were presented as a function of their concentrations in Fig. 1. The DC*H+ and Clelectrodes showed a linear response with a Nemstian slope at concentrations below 20.0 mmol kg-^ Both electrode responses deviated from the Nemstian slope at concentrations above 20.0 mmol kg-^: the depression of activities for both ions was observed. At concentrations above 80.0 mmol kg->, the activity of DC»H+ had a constant value and the slope of activity of CIbecame about one-third of Nemstian response. Since the CMC of DC^HCl has a value of 78.7 mmol kg-' [6] and no hydrolysis of DC»H+ occurred in this study [5], we examined the activity depressions taking account of premicellar formation due to self-association of E>C»HC1 in the aqueous solution. Because the activities of DC»H+ and CI" were depressed each other, it is expected that premicelles including both ions are formed in the solution. We consider that other monovalent cationic species except DC»H+ are able to response the DC^H+ electrode as follows -3.0
-2.5
-2.0
-1.5
-1.0
log [DC-HCI],. log [NaCI] / mol dm^
mDC-H"*" + (m - l)Cr
Fig. 1. Relationship between electrode potential and E)C»HC1 or NaCl concentration: The equilibrium constant {K) of the above reaction (1) dibucaine cation, (2) chloride ion is expressed as (DC*HC1), (3) chloride ion (NaCl). -(DC-H)^Cl(,.,/
(1)
115 K = [(DC*H)^Cl(^.,)^]/[DC.H*r[Cr]^'"-^^
(2)
The apparent concentration of DC • H+ ([DC • H"^\^^) obtained from the electrode is given by iapp>' [DC-H^],pp = [DC-H*] + A [ ( D C - H ) , a ( , . , ) n
(3)
where X is the response coefficient of the electrode for (DC»H)^C1(^. ^"^ ion. Total concentration of dibucaine ([IX: • HClJt) can be written using the concentration of DC-H+ [DC*HC11^ = [DC*H^] + m[(DC*H)^Cl(^.,)^]
(4)
From Eqs. (3) and (4), we obtain [DC-HCl], - [DC-H^],pp = (m -A)[(DC-H)^C1(,. ,)^]
(5)
Alternatively, using CI" ion concentration, [DC* HClj^ is written as [DC*HCl]t - [CI] = (m .1)[(DC*H)^C1(^.,)^]
(6)
By combining Eqs. (5) and (6), ([DC*HCl]t - [DCH^],pp)/([DC•HCIJj - [CI]) = (m - ^)/(m - 1)
(7)
The A value can be determined experimentally if we can know the m value. Considering ionic equilibrium given by Eq. (1), the following two equations can be obtained. ([DC*HCl]t - [DC•H-'])/[DC•H^]'" = mA:[Cr]^'" ''^
(8)
and ([DC*HC1], - [Cr])/[Cl-]^'"-^> = (m -1)A:[DC«H^]'"
(9)
where [DC • H"*"] is not measurable directly and obtainable from the equation [DC*H^] = [DC-H^],pp - A/(m -l)([DC-HCI]t - [Cl'l)
(10)
Right hand side of Eq. (10) can be determined with the m value by the EMF measurements. Both activity depressions were analyzed by the above equations with several m values. We found that the m value of 3 held on the Eqs. (8) and (9). The results are shown in Fig. 2,
10
20
(Crf X I0*/mol^dm*
20
40
60
[DC«H*f X I0*/mol^clm'"
Fig. 2. Validity of pre-micelle formation with m = 3: (A) plot of Eq. (8), (B) plot of Eq. (9).
116 respectively: good linear relationship in both figures with m = 3 was obtained. The values of ^ and A were found to be 6.21 (± 0.18) x 10* (moH kg^) and 0.50 ± 0.14 at 298.15 K, respectively. This fact indicates that at the concentration below the CMC, DC^HCl forms a trimer with two chloride ions. The pre-micelle formation of dibucaine may be attributable to the n electron interaction of an aromatic ring with a butyl chain in the molecule. 3.2. Formation and growth of DC*HC1 micelle at concentrations above the CMC We next performed the static and dynamic light scattering (SLS and DLS) measurements on various concentrations of aqueous E>C»HC1 solutions in the absence and presence of added sodium chloride (NaCl). The static scattered light intensity greatly increased at concentrations above the CMC. The aggregation numbers of the anesthetic micelle were evaluated from Debye plot of the intensity data. The value of 15 was obtained for DC»HC1 micelles in water and it is consistent with literature ones [3,4]. Since E)C»HC1 form trimers at concentrations below the CMC, five dibucaine trimers associate cooperatively and form the micelle at concentrations above the CMC. Furthermore, the CMC decreased and the aggregation number increased by the addition of NaCl. Micellar properties of E)C»HC1 in water and NaCl solutions were sununarized in Table 1 together with the average diameters of DC^HCl micelle, which were obtained by DLS measurements. Although the average diameter increased with increasing NaCl concentration, the variation of the aggregation number with NaCl concentration seemed not to be threedimensional as seen in surfactants with a straight hydrophobic chain. This fact suggests that the E>C»HC1 micelle grows one-dimensional direction like small rod-like micelle in the aqueous solution by addition of NaCl. The micellar growth of DC*HC1 in solutions of high ionic strength may result from the stacking of an aromatic ring in the molecule. Table 1 Micellar properties of DC*HC1 in water and NaCl solutions NaCl cone. (molkg-»)
0
0.10
0.15 a)
0.30
0.40
0.50
0.60
79.4
51.0
40.9
28.2
22.5
20.8
18.3
Aggregation number
15
29
34
40
45
53
59
Average diameter (nm)
1.9
2.8
3.3
4.1
4.8
5.2
6.1
CMC(mmolkg-0
*) physiological saline concentration. REFERENCES 1. T. Eckert, E. Kilb and H. Hoffman, Arch. Pharm., 297 (1964) 31. 2. R. Jaenicke, Kolloid. Z., 212 (1966) 36. 3. E. H. Johnson and D. B. Ludlum, Biochem. Pharmacol., 18 (1969) 2675. 4. D.Attwood and P Fletcher, J. Pharm. Pharmacol., 38 (1986) 494. 5. H. Matsuki and S. Kaneshina, Hyomen (in Japanese), 37 (1999) 20. 6. H. Matsuki, M. Yamanaka, S. Kaneshina, H. Kamaya and I. Ueda, Colloids Surfaces B: Biointerfaces, 11 (1998) 87. 7. H.Satake, T. Miyata and S. Kaneshina, Bull. Chem. Soc. Jpn., 64 (1991) 3029.
113
Pre-micelle and micelle formation of local anesthetic dibucaine hydrochloride Hitoshi Matsukia, Takahiro Miyata^, Toshiharu Yoshioka^, Hiromu Satake*> and Shoji Kaneshinaa ^Department of Biological Science and Technology, Faculty of Engineering, and ^'Center for Cooperative Research, The University of Tokushima, Minamijosanjima, Tokushima 770-8506, Japan The molecular-aggregate formation of local anesthetic dibucaine hydrochloride (E)C»HC1) was investigated from the electrode potential and light scattering measurements of the aqueous solutions. The electrode potential of E)C»HC1 solutions showed the deviation from Nemstian response at low concentrations below the critical micelle concentration (CMC). We considered the possibility of pre-micelle formation of DC»HC1 in the solution and found that dibucaine cation forms a trimer with two chloride ions in the concentration range. On the other hand, the aggregation number of 15 were obtained for DC^HCl micelles in water from the light scattering measurements. Five dibucaine trimers associate cooperatively and form the micelle at concentrations above the CMC. Further, we observed the E)C»HC1 micelle grow one-dimensional direction like small rod-hke micelle in solutions of high ionic strength. The pre-micelle, micelle formation and micellar growth of dibucaine may be attributable to the stacking of dibucaine molecules due to the ;r electron interaction of an aromatic ring in the molecule. 1. INTRODUCTION Local anesthetics have tertiary amine structures with an aromatic ring. Some with large hydrophobicities form molecular aggregates such as micelles in aqueous solutions. The potency of local anesthetics to block nerve conduction is based on their ability to associate with membranes. There are numerous reports demonstrating that the clinical potency of local anesthetics correlates extremely well with their ability to adsorb to lipid monolayers and bilayers. The formation of molecular aggregates and the partitioning into hydrophobic environments of cell membranes, which is an important process for local anesthesia, are both based on the hydrophobicity. The micelle formation of local anesthetics in aqueous solutions has beenreportedby several researchers [1-4]. They showed that the aggregation of local anesthetics is different from that of surfactants with a straight hydrophobic chain. Recently, we have also shown the existence of micellar aggregates for local anesthetics in the aqueous solution by ionic activity, differential scanning calorimetry (DSC), density, and surface tension measurements [5], etc. In the present study, we examined the formation of pre-micellar and micellar aggregates for dibucaine hydrochlorides (DC»HC1) from the electrode potenrial and light scattering measurements of the aqueous solutions. The aggregation behavior of DC^HCl in the aqueous solution at concentrations below and above the critical micelle concentration (CMC) and that in solutions of high ionic strength was characterized.
114 2. EXPERIMENTAL Dibucaine hydrochloride (2-butoxy-N-[2-(diethylamino)ethyl]-4-quinolinecarboxamide hydrochloride: C2oH29N302»HCl) was purchased from Sigma Chemicals (St. Louis, MO) and purified by the method described previously [6]. Water was distilled twice after deionization. The coated-wire electrode selectively sensitive to dibucaine cation was prepared by the same method described previously [7]. The electromotive force (EMF) of the dibucaine cation and chloride ion (Toko Chemicals' electrodes 7020) was measured by a digital multi-ion monitor (Yamashita Giken Co. Ltd.,Tokushima, Japan) at 298.15 K under atmospheric pressure [7]. Static and dynamic light scattering measurements were performed by light-scattering spectrophotometer DLS-7000 (Otsuka Electronics, Osaka, Japan) with Ar and He-Ne lasers. The scattered light of aqueous dibucaine solutions in the absence and presence of sodium chloride was measured under the same condition as the EMF measurements. The increment of refractive index for dibucaine solutions was measured by differential refractometer DRM-1021 (Otsuka Electronics) to calculate the weight average of molecular weight for dibucaine. 3. RESULTS AND DISCUSSION 3.1. Pre-micelle formation of DC-HCI at concentrations below the CMC The electrode responses of dibucaine cation (DC»H+) and chloride ion (CI) in the aqueous solution were presented as a function of their concentrations in Fig. 1. The DC*H+ and Clelectrodes showed a linear response with a Nemstian slope at concentrations below 20.0 mmol kg-^ Both electrode responses deviated from the Nemstian slope at concentrations above 20.0 mmol kg-^: the depression of activities for both ions was observed. At concentrations above 80.0 mmol kg->, the activity of DC»H+ had a constant value and the slope of activity of CIbecame about one-third of Nemstian response. Since the CMC of DC^HCl has a value of 78.7 mmol kg-' [6] and no hydrolysis of DC»H+ occurred in this study [5], we examined the activity depressions taking account of premicellar formation due to self-association of E>C»HC1 in the aqueous solution. Because the activities of DC»H+ and CI" were depressed each other, it is expected that premicelles including both ions are formed in the solution. We consider that other monovalent cationic species except DC»H+ are able to response the DC^H+ electrode as follows -3.0
-2.5
-2.0
-1.5
-1.0
log [DC-HCI],. log [NaCI] / mol dm^
mDC-H"*" + (m - l)Cr
Fig. 1. Relationship between electrode potential and E)C»HC1 or NaCl concentration: The equilibrium constant {K) of the above reaction (1) dibucaine cation, (2) chloride ion is expressed as (DC*HC1), (3) chloride ion (NaCl). -(DC-H)^Cl(,.,/
(1)
115 K = [(DC*H)^Cl(^.,)^]/[DC.H*r[Cr]^'"-^^
(2)
The apparent concentration of DC • H+ ([DC • H"^\^^) obtained from the electrode is given by iapp>' [DC-H^],pp = [DC-H*] + A [ ( D C - H ) , a ( , . , ) n
(3)
where X is the response coefficient of the electrode for (DC»H)^C1(^. ^"^ ion. Total concentration of dibucaine ([IX: • HClJt) can be written using the concentration of DC-H+ [DC*HC11^ = [DC*H^] + m[(DC*H)^Cl(^.,)^]
(4)
From Eqs. (3) and (4), we obtain [DC-HCl], - [DC-H^],pp = (m -A)[(DC-H)^C1(,. ,)^]
(5)
Alternatively, using CI" ion concentration, [DC* HClj^ is written as [DC*HCl]t - [CI] = (m .1)[(DC*H)^C1(^.,)^]
(6)
By combining Eqs. (5) and (6), ([DC*HCl]t - [DCH^],pp)/([DC•HCIJj - [CI]) = (m - ^)/(m - 1)
(7)
The A value can be determined experimentally if we can know the m value. Considering ionic equilibrium given by Eq. (1), the following two equations can be obtained. ([DC*HCl]t - [DC•H-'])/[DC•H^]'" = mA:[Cr]^'" ''^
(8)
and ([DC*HC1], - [Cr])/[Cl-]^'"-^> = (m -1)A:[DC«H^]'"
(9)
where [DC • H"*"] is not measurable directly and obtainable from the equation [DC*H^] = [DC-H^],pp - A/(m -l)([DC-HCI]t - [Cl'l)
(10)
Right hand side of Eq. (10) can be determined with the m value by the EMF measurements. Both activity depressions were analyzed by the above equations with several m values. We found that the m value of 3 held on the Eqs. (8) and (9). The results are shown in Fig. 2,
10
20
(Crf X I0*/mol^dm*
20
40
60
[DC«H*f X I0*/mol^clm'"
Fig. 2. Validity of pre-micelle formation with m = 3: (A) plot of Eq. (8), (B) plot of Eq. (9).
116 respectively: good linear relationship in both figures with m = 3 was obtained. The values of ^ and A were found to be 6.21 (± 0.18) x 10* (moH kg^) and 0.50 ± 0.14 at 298.15 K, respectively. This fact indicates that at the concentration below the CMC, DC^HCl forms a trimer with two chloride ions. The pre-micelle formation of dibucaine may be attributable to the n electron interaction of an aromatic ring with a butyl chain in the molecule. 3.2. Formation and growth of DC*HC1 micelle at concentrations above the CMC We next performed the static and dynamic light scattering (SLS and DLS) measurements on various concentrations of aqueous E>C»HC1 solutions in the absence and presence of added sodium chloride (NaCl). The static scattered light intensity greatly increased at concentrations above the CMC. The aggregation numbers of the anesthetic micelle were evaluated from Debye plot of the intensity data. The value of 15 was obtained for DC»HC1 micelles in water and it is consistent with literature ones [3,4]. Since E)C»HC1 form trimers at concentrations below the CMC, five dibucaine trimers associate cooperatively and form the micelle at concentrations above the CMC. Furthermore, the CMC decreased and the aggregation number increased by the addition of NaCl. Micellar properties of E)C»HC1 in water and NaCl solutions were sununarized in Table 1 together with the average diameters of DC^HCl micelle, which were obtained by DLS measurements. Although the average diameter increased with increasing NaCl concentration, the variation of the aggregation number with NaCl concentration seemed not to be threedimensional as seen in surfactants with a straight hydrophobic chain. This fact suggests that the E>C»HC1 micelle grows one-dimensional direction like small rod-like micelle in the aqueous solution by addition of NaCl. The micellar growth of DC*HC1 in solutions of high ionic strength may result from the stacking of an aromatic ring in the molecule. Table 1 Micellar properties of DC*HC1 in water and NaCl solutions NaCl cone. (molkg-»)
0
0.10
0.15 a)
0.30
0.40
0.50
0.60
79.4
51.0
40.9
28.2
22.5
20.8
18.3
Aggregation number
15
29
34
40
45
53
59
Average diameter (nm)
1.9
2.8
3.3
4.1
4.8
5.2
6.1
CMC(mmolkg-0
*) physiological saline concentration. REFERENCES 1. T. Eckert, E. Kilb and H. Hoffman, Arch. Pharm., 297 (1964) 31. 2. R. Jaenicke, Kolloid. Z., 212 (1966) 36. 3. E. H. Johnson and D. B. Ludlum, Biochem. Pharmacol., 18 (1969) 2675. 4. D.Attwood and P Fletcher, J. Pharm. Pharmacol., 38 (1986) 494. 5. H. Matsuki and S. Kaneshina, Hyomen (in Japanese), 37 (1999) 20. 6. H. Matsuki, M. Yamanaka, S. Kaneshina, H. Kamaya and I. Ueda, Colloids Surfaces B: Biointerfaces, 11 (1998) 87. 7. H.Satake, T. Miyata and S. Kaneshina, Bull. Chem. Soc. Jpn., 64 (1991) 3029.