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Lidocaine transport through a cellophane membrane by alternating current iontophoresis with a duty cycle
Bioelectrochemistry 74 (2009) 315–322
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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m
Lidocaine transport through a cellophane membrane by alternating current iontophoresis with a duty cycle Shizuka Hayashi a,⁎, Saori Ogami a, Takao Shibaji b, Masahiro Umino a a Section of Anesthesiology and Clinical Physiology, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Section of Orofacial Pain Management, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
a r t i c l e
i n f o
Article history: Received 28 March 2008 Received in revised form 12 November 2008 Accepted 18 November 2008 Available online 30 November 2008 Keywords: Iontophoresis Alternating current Lidocaine Duty Cycle Cellophane membrane
a b s t r a c t The purpose of this study was to determine whether lidocaine can be efficiently transported across a cellophane membrane using a square-wave alternating current (AC) with an adjusted duty cycle. Three voltages at 1 kHz with 6 duty cycles were applied for 60 min to the diffusion cells on both sides of the cellophane membrane. The donor chamber was filled with 1% lidocaine hydrochloride solution. The transport of lidocaine was enhanced in a voltage-, and duty cycle-dependent manner. These findings indicate that voltage and the direct current (DC) component of the square-wave AC play important roles in generating the driving force necessary for lidocaine delivery. Additionally, the periodic polarity alteration could reduce the electrode polarization. The higher voltages and duty cycles induced a pH change. The practical electrical conditions which are preferable for clinical application were 10 V with a 70% duty cycle or 20 V with a 60% duty cycle. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Iontophoresis is a delivery method by which the movement of ionic compounds or non-charged drug molecules is affected by the application of an external electric field. Iontophoresis enhances the transport of ionic substances across membranes or the skin via three principal mechanisms: electrorepulsion, electroosmosis and electroporation. Among these, electrorepulsion is considered to play the most crucial role in the transport of ionic drugs. Transdermal drug delivery using iontophoresis has a number of potential advantages, e.g., drug degradation due to passage through the stomach or intestine or the first-pass through the liver is avoided, it is user-friendly, and it is a painless method. Transdermal iontophoresis using a direct current (DC) has been used for both local and systemic drug delivery. Applications include local delivery of anesthetics, steroids and retinoids to treat scarring caused by acne, for the relief of palmar and plantar hyperhidrosis [1], and iontophoresis of fentanyl is also used for the management of postoperative pain or cancer pain [2]. However, some adverse effects, including electrical burns or erythema as a result of electrode polarization during electrolysis, have been reported in clinical situations [3]. This negative side effect limits the application time of DC iontophoresis to less than 15 min at current densities as low as 1 mA/cm2 [4]. Furthermore, the transport efficiency is also reduced with increasing duration of the electrical application. The lowered transport efficiency is caused by a ⁎ Corresponding author. Tel.: +81 3 5803 5549; fax: +81 3 5803 0206. E-mail address: [email protected] (S. Hayashi). 1567-5394/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2008.11.007
voltage drop in the solution resulting from the formation of an electric double layer on the electrode surface (a phenomenon known as ‘electrode polarisation’), because of the accumulation of ionized substances with a different charge from that of the electrode polarity. To resolve this problem, alternating current (AC) iontophoresis has also been studied. Howard et al. reported that the AC-iontophoretic transport of hydroxocobalamin through human skin was more efficient than passive transport [5]. Yan and Peck et al. applied square-wave AC iontophoresis across a synthetic membrane and confirmed that flux of tetraethyl ammonium and arabinose were enhanced by increasing the voltage and decreasing the frequency of the AC [6]. We have also studied ion transport using AC. Shibaji et al. revealed that physiological saline could be transported through a cellophane membrane using a sinusoidal AC voltage at 1 kHz [7]. Izumikawa reported transport of lidocaine through a cellophane membrane using AC, and the transport efficiency was markedly enhanced at 1 kHz [8]. Kinoshita et al. found that lidocaine could be transported through excised rat skin in vitro using a sinusoidal AC voltage [9]. We succeeded in transport of lidocaine through living rat skin using AC in a voltage- and time-dependent manner [10]. Furthermore, Yan and Peck et al. reported that AC with a DC offset voltage enhances the transport of both neutral and ionic permeants [6]. On the other hand, Reinauer et al. found no effect of the application of sawtooth wave AC iontophoresis for the treatment of palmar hyperhidrosis [11]. These reports suggest that AC with a DC component may be useful for the transport of ionic compounds. The degree of electrode polarization on the electrode or skin surface in the case of application of AC is less than that in the case of DC,
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because of the periodic polarity alteration. Accordingly, the decrease of the transport efficiency of ionic compounds with increasing time is alleviated. However, the transport efficiency of ionic compounds by electrorepulsion in AC is inferior to that by DC, because AC has no direct current component. If AC is combined with a DC component, electrorepulsion due to the DC component can be expected despite the periodic polarity alteration. Therefore, we applied a bipolar square wave with a duty cycle for lidocaine transport across the cellophane membrane. The purpose of this study was to determine whether lidocaine can be efficiently transported across a cellophane membrane using a square-wave AC with an adjusted duty cycle, and to find the optimal duty cycle and voltage for such lidocaine transport. 2. Materials and methods 2.1. Materials Lidocaine hydrochloride (C14H22N2O∙HCl, FW: 270.8, H2O content: 1 mol/mol) was purchased from Sigma-Aldrich Co. Ltd. (St. Louis, USA). One percent lidocaine hydrochloride was prepared using distilled and deionized water (resistivity N18 MΩ q cm). The pH of the 1% lidocaine hydrochloride solution was 4.6. The solution was degassed using an ultrasonic cleaner for 30 min immediately before injection into the chamber. 2.2. Membrane The cellophane membrane (Futamura Chemical Co., Ltd., Nagoya, Japan) was about 36 μm thick, with a pore size of about 2–3 nm. This pore size was about twofold larger than the size of the lidocaine molecules. 2.3. Experimental cell and transport system A cylindrical acryl drug delivery cell consisting of two chambers was originally constructed. Platinum plate electrodes with a diameter of 20 mm and thickness of 0.2 mm were installed at opposite ends of the two components of the cell. The length of each component was 10 mm (Fig. 1). The cellophane membrane was sandwiched between the two components (one of which was the donor chamber
and the other, the receptor chamber). The available diffusion area was about 3.1 cm2 and the capacity of the each chamber was about 3.0 ml. The donor chamber was filled with 1% lidocaine hydrochloride solution (3.0 ml, pH 4.6) and the receptor chamber with distilled water (3.0 ml). The acryl cells were set in the water bath, and the temperature of the cell was controlled so as to not allow it to exceed 36.5 °C. A thermocouple microprobe (BAT-12, Physitemp, NJ, USA) was inserted at the center of the donor chamber to monitor the temperature of the solution contained in it. The solution in the two cell chambers was not stirred, because the bulk flow caused by stirring and the rotator-induced magnetic field can affect lidocaine transport. The aqueous boundary layer due to the absence of stirring in the cell was negligible. Constant-voltage AC was continuously applied between the parallel platinum electrodes for 60 min using a function/arbitrary waveform generator (Agilent 33250A, Agilent Technologies, Colorado, USA) and a high speed power amplifier (4025, NF Electric Instruments, Kanagawa, Japan), and the waveform and voltage of output were monitored with a digitizing oscilloscope (HP54503A, Hewlett Packard, Tokyo, Japan) throughout the duration of the experiment. 2.4. Determination of the lidocaine concentration and pH Twenty μl of solution was sampled every 10 min after the start of the AC application for 60 min, with a micropipette (20–200 μl, Nichiryo, Tokyo, Japan) placed at the center of the receptor chamber. The concentration of lidocaine in the receptor chamber was determined using a spectrophotometer (U-3310; absorbance range: −2∼4 Abs; precision: ±0.002 Abs, HITACHI, Tokyo, Japan) at room temperature. Before the analysis, the samples were diluted 40 times with distilled and deionized water. The absorbance values of the samples were measured at a wavelength of 262 nm and optical path of 10 mm. The concentration of lidocaine was quantified using a calibration curve. The detection limit was 11.6 μg/ml. The pH of the samples was measured with an FET (Field Effect Transistor) pH meter (pH BOY-P2, Shindengen Electric MFG Co., Tokyo, Japan). The efficiency of iontophoresis was defined as the slope of the timecourse of increase in the transport of lidocaine from the donor chamber to the receptor chamber during the experiment. The efficiency of
Fig. 1. The setup of the experimental system. The experimental system consists of two chambers, a thermocouple microprobe, a water bath, a function generator and a high speed power amplifier.
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2.6. Statistical analysis All values are presented as mean ± S.D. (n = 5). A two-way ANOVA test was used to analyze the time- and duty cycle-dependences of the transport efficiency of lidocaine. In addition, the Tukey test was used to analyze the duty cycle- and voltage-dependences of the transport
Fig. 2. Diagram of a square-wave AC with a 100a% duty cycle (a = 0.5, 0.6, 0.7, 0.8, 0.9, 0.95). T is the period of the square wave. A 100a% duty cycle represents the ratio of the positive cycle to the full cycle. The ratio of the positive cycle was adjusted between 50%, 60%, 70%, 80%, 90% and 95%.
iontophoresis was calculated by applying the least-square method, expressed by Eq. (1) as follows: 6
2
y = ax + b; r 2 = ∑ ½yi −ðaxi + bÞ ; i=1
Ar 2 Ar 2 = 0; =0 Aa Ab
ð1Þ
where xi (i = 1, …, 6) represents the time [min] (x1 = 10, x2 = 20, …, x6 = 60), yi is the concentration of lidocaine in the receptor chamber [μmol/cm3], a is the slope of the time-course of increase in the transport of lidocaine [μmol/cm3/min], b is a constant, and r is the correlation coefficient. The variable a is defined as the efficiency of iontophoresis, which is calculated by the gradient of the approximate linear equation fitting the time-course curves for ion concentration in the receptor chamber during AC iontophoresis. In addition, the flux [μmol/h/cm2] of lidocaine through cellophane membrane was calculated from the cumulative amount of lidocaine transported to the receptor chamber over a period of 60 min. 2.5. Experimental design To examine the relationship between the efficiency of lidocaine transport and the duty cycle, a bipolar square wave with 6 different duty cycles, that is, 50%, 60%, 70%, 80%, 90% and 95%, was continuously applied at 1 kHz for 60 min to the drug delivery cell, with the donor chamber containing 1% lidocaine solution, under 3 different constant voltages of 10, 20 and 30 V, using a function/arbitrary waveform generator (Agilent 33250A, Agilent Technologies, Colorado, USA) and a high speed power amplifier (4025, NF Electric Instruments, Kanagawa, Japan) (Fig. 2). The lidocaine concentration in the receptor chamber was determined by spectrophotometry (U-3310; absorbance range: −2∼4 Abs; precision: ±0.002 Abs, HITACHI, Tokyo, Japan) at 10, 20, 30, 40, 50 and 60 min after the start of the current application. The pH of the solution in the receptor chamber was measured with an FET pH meter (pH BOY-P2, Shindengen Electric MFG Co., Tokyo, Japan) at 10, 20, 30, 40, 50 and 60 min after the start of the current application for each duty cycle and voltage condition.
Fig. 3. Changes of the lidocaine concentrations for applied a square-wave AC with 6 different duty cycles under (a) 10 V, (b) 20 V and (c) 30 V at 1 kHz: (○) passive, (●) duty cycle 50%, (□) 60%, (■) 70%, (△) 80%, (▲) 90%, (▽) 95%. The error bars represent standard deviation of the mean. The lidocaine concentrations increased significantly in a time-dependent and approximately linear manner for every duty cycle.
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efficiency of lidocaine and the relationship between the duty cycle and pH. Statistical significance was set at p b 0.05. 3. Results
On the other hand, the pH of the solution in the donor chamber declined in a duty cycle-dependent manner at 10, 20 and 30 V. With the 95% duty cycle at 10 V and the 80%, 90%, and 95% duty cycles at 20 V and 30 V that were applied for 60 min, the pH of the donor chamber was about 1.8.
3.1. Lidocaine concentration and duty cycles 4. Discussion Fig. 3a–c shows the changes of the lidocaine concentrations in the receptor chamber induced by the application of a square-wave AC with 6 different duty cycles, that is, 50%, 60%, 70%, 80%, 90% and 95%, under 10, 20 or 30 V at 1 kHz. The lidocaine concentrations in the receptor chamber increased significantly in a timedependent and approximately linear manner for every duty cycle under 10 V (F = 5.49, p b 0.001; Fig. 3a), 20 V (F = 5.33, p b 0.001; Fig. 3b) and 30 V (F = 8.75, p b 0.001; Fig. 3c). The lidocaine concentration obtained with application of the 95% duty cycle at 30 V was about twofold higher than that obtained with application of the 50% duty cycle, and about threefold higher than that observed following passive diffusion, at 60 min after the start of the current application (Fig. 3c). Fig. 4a–c shows the lidocaine transport efficiency obtained for the 6 different duty cycles under 10 V (Fig. 4a), 20 V (Fig. 4b) and 30 V (Fig. 4c) at 1 kHz. The lidocaine transport efficiency increased in a linear manner as the duty cycle increased, at each of the 3 voltages used. At 10 and 20 V, the lidocaine transport efficiencies with the application of 70%, 80%, 90% and 95% duty cycles were significantly higher as compared with the efficiency obtained with a 50% duty cycle (p b 0.05; Fig. 4a–b). At 30 V, the lidocaine transport efficiency with the application of duty cycles of 60%, 70%, 80%, 90% and 95% were significantly higher compared with that observed with a 50% duty cycle (p b 0.001; Fig. 4c). 3.2. Lidocaine transport efficiency and voltages At the duty cycle of 50%, the lidocaine transport efficiency increased with increase of voltage, however, there were no significant differences in the lidocaine transport efficiency among the three voltages examined. Although the lidocaine transport efficiency at 30 V was significantly higher than that at 10 and 20 V (p b 0.05) for the duty cycles of 60%, 70%, 80% and 90%, no significant differences in the lidocaine transport efficiency were observed between 10 and 20 V for any width of the duty cycles. At the duty cycle of 95%, the lidocaine transport efficiency at 30 V was significantly higher than that at 20 V, and that at 20 V was significantly higher than the efficiency at 10 V (p b 0.05). Fig. 5 shows the relationship between the voltage and the flux of lidocaine. The lidocaine flux depended on the applied voltage. At the duty cycle of 95%, the average flux under the application of 30 V for 60 min was about 1.3-fold higher than that obtained with application of 10 V, and about threefold higher than that observed following passive diffusion.
4.1. Square-wave AC and lidocaine transport efficiency The present study revealed that application of a square-wave AC with a duty cycle, which has both components of direct and alternating currents, was effective for accomplishing successful transport of lidocaine in a time-, voltage-, and duty cycle-dependent manner. These findings indicate that the voltage and the DC component of the square-wave AC play important roles in generating the driving force necessary for lidocaine delivery. Additionally, the periodic polarity alteration could reduce the electrode polarization, which disturbs the transport of lidocaine ions, resulting in enhancement of the lidocaine transport efficiency. Application of an electric field causes a polarization of the skin or the electrode surface oriented in the direction opposite to the applied field. Marked electrode polarization occurs with the application of DC alone. The effective current diminishes with increase of the DC application time, because electrode polarization of the skin and the electrode surface act as a capacitor in an electric circuit. Polarization lowers the transport efficiency of substances. Until now, various waveforms have been used to avoid polarization of the electrode surface and skin in iontophoresis. Iontophoresis using AC in combination with DC has also been attempted for the treatment of palmoplantar hyperhidrosis, and was shown to be associated with reduced side effects [11]. Pulsed DC with duty cycles ranging from 20% to 80% has been studied both at the basic and clinical levels [11–13]. To decrease the electrode polarization, we have previously attempted application of a sine-wave AC at various frequencies and voltages for lidocaine transport, both in vitro and in vivo [8–10]. However, the driving force with the use of a sine-wave AC is inferior to that with the application of DC, because of the periodic polarity alteration. Therefore, a square wave with polarity alteration (square-wave AC) was employed in the present study. In the iontophoretic study, artificial membranes, animal or human skins have been used as a partition placed between donor and receptor cells. In the present study, cellophane membrane was employed because it has not been investigated whether ionized substances can be transported through an artificial partition by a square wave with polarity alteration. The AC component with the periodic polarity alteration generates less electrode polarization and the square wave generates the driving force for the drug. The average voltage Uave for one period of the square wave can be expressed as 1 T ∫ U ðt Þdt T 0
ð2Þ
3.3. Duty cycle and pH change
Uave =
Table 1 shows the pH of the solution in the receptor chamber for the application of a square-wave AC with 6 different duty cycles, that is, 50%, 60%, 70%, 80%, 90% and 95% under 10, 20 and 30 V, at 1 kHz. The pH of the solution in the receptor chamber increased in a duty cycle-dependent manner at 10, 20 and 30 V. The pH declined in the case of passive diffusion and with the application of a 50% duty cycle, at 10, 20 and 30 V. There were no significant differences in the pH between duty cycles of 50% and 60% at 10 V. The pH following application of duty cycles of 70%, 80%, 90% and 95% was significantly higher than that observed with a 50% duty cycle at 10 V (p b 0.05). The pH following the application of duty cycles of 60%, 70%, 80%, 90% and 95% was significantly higher than that observed with the application of a 50% duty cycle at 20 V and 30 V (p b 0.05).
where t is the time, U(t) is the voltage between the electrodes at t, and T is the period of the square wave. When a constant AC voltage A with a 100a (%) duty cycle was applied, the voltage between the electrodes is A from 0 (s) to aT (s) after the start of the current application, and –A from aT (s) to T (s) (Fig. 2). Therefore, Eq. (2) can be reduced to Uave =
o 1 n aT T ∫ 0 Adt + ∫aT ð−AÞdt T = ð2a−1ÞA
ð3Þ
where a is the duty cycle (a = 0.5, 0.6, 0.7, 0.8, 0.9, 0.95), and A is the amplitude of the square-wave AC. From Eq. (3), it can be seen that Uave
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Fig. 4. The change of the lidocaine transport efficiency as a function of the duty cycle of square-wave AC under (a) 10 V, (b) 20 V and (c) 30 V at 1 kHz. The efficiency of lidocaine transport increased in a linear manner as the duty cycle increased, at each of the 3 voltages.
remains constant with respect to time when voltage and duty cycle are constant. And Uave can be regarded as the DC component and effective for penetration of drug. From Eq. (3), it is seen that Uave is a linear function of the duty cycle when a constant AC voltage is applied, and
when the duty cycle is constant, the Uave is proportional to the voltage between the electrodes. In the present study, the lidocaine transport efficiency showed voltage dependence at all the duty cycles applied. This finding indicates that
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Fig. 5. The change of the lidocaine flux as a function of the applied voltage when a 95% duty cycle at 1 kHz was applied. The flux of lidocaine increased in a voltage-dependent manner.
the voltage is closely related to the driving force. Our previous in-vivo and in-vitro studies using the sine-wave AC also revealed voltage dependence of lidocaine transport [8,9]. The frequency of the current and the degree of ionization of a substance are also known to influence the transport efficiency. In our study, the frequency of 1 kHz was employed, because our previous study results showed that lidocaine can be effectively transported at this frequency [9]. A 1% lidocaine solution was used in the present study. Lidocaine, with a molecular weight of 234.3, is composed of a lipophilic part (a benzene ring), a hydrophilic part (a tertiary amine), and an intermediate carboxy linkage. For commercial use, lidocaine is used as its hydrochloride salt. Lidocaine hydrochloride (salt) is quite soluble in water. In aqueous solution, the lidocaine hydrochloride molecule dissociates and ionizes to yield a quaternary amine cation plus an acid anion. In the present study, the pH of the lidocaine solution in the donor chamber ranged from 4.6 to 1.8. Since the pKa of lidocaine is 7.86, the lidocaine molecules were almost completely dissociated and ionized in the aforementioned pH range. Lidocaine molecules dissociated into cation were transported to the receptor chamber by the square-wave AC.
Drug ion transport is a function of the total current and the fluxes of other ions present. The transport rates of other species are also dependent upon the total current. The donor chamber used in the present study contained positively charged lidocaine ions and negatively charged chloride ions. The ion current was produced by movement of the lidocaine ions as well as that of the other ions in the donor chamber with the AC application [14]. The transport rate of a drug depends on its transference number (the fraction of the current carried by the drug ion) and the total current driven, which in turn, is related to the voltage drop across the rate-limiting resistance to transport of the drug [14]. Although the lidocaine transport efficiency was the most effective with the use of the 95% duty cycle at 30 V in the present study, the pH of the solution in the receptor chamber was markedly increased under this condition because of water electrolysis. Application of a duty cycle of 70% at 10 V or 60% at 20 V produced effective transport as compared with that observed with the use of a 50% duty cycle, with a smaller change of the pH of the solution in the receptor chamber. These results suggest that application of a duty cycle of 70% at 10 V or 60% at 20 V is preferable for clinical application, because of the less marked occurrence of water electrolysis as compared with that observed with a 95% duty cycle at 30 V. 4.2. Transport mechanism The present study demonstrated that the transport efficiency of lidocaine depended on the duty cycle at each of the AC voltages examined. This finding shows that the width of the DC component in the square wave was closely associated with the driving force of lidocaine. The mole flux of substance i, which is named Ji, is defined as Ji = ðdni =dt Þ=A
ð4Þ
where ni is the number of substance i and A the transporting area. Although the transport mechanism of substances driven by the application of AC with a duty cycle has not yet been completely elucidated, the following Eq. (5) may explain the substance transport mechanism with the application of an external electric field [13,15]. ð5Þ
Ji = Jp + Jer + Jeo
where Jp is the passive flux, Jer is the electrorepulsive contribution, in which the ion is repelled from an electrode of the same charge, and Jeo
Table 1 The pH changes of the solution in the receptor chamber Applied voltage
Duty cycle
t (min) 0
10
20
30
40
50
60
10 V
50% 60% 70% 80% 90% 95% 50% 60% 70% 80% 90% 95% 50% 60% 70% 80% 90% 95%
6.4 ± 0.00 6.5 ± 0.02 6.4 ± 0.02 6.5 ± 0.01 6.5 ± 0.06 6.4 ± 0.02 6.4 ± 0.00 6.4 ± 0.07 6.4 ± 0.01 6.4 ± 0.04 6.5 ± 0.06 6.5 ± 0.07 6.4 ± 0.21 6.3 ± 0.25 6.4 ± 0.17 6.4 ± 0.10 6.4 ± 0.12 6.4 ± 0.06 6.1 ± 0.00
5.4 ± 0.28 6.4 ± 0.15 7.8 ± 0.42 * 7.8 ± 0.26 * 8.4 ± 0.56 * 8.8 ± 0.15 * 5.7 ± 0.00 7.6 ± 1.20 * 8.1 ± 0.24 * 8.6 ± 0.49 * 8.8 ± 0.01 * 9.4 ± 0.00 * 5.7 ± 0.00 8.2 ± 0.15 * 8.9 ± 0.10 * 8.8 ± 0.15 * 9.5 ± 0.15 * 9.3 ± 0.17 * 6.0 ± 0.06
5.1 ± 0.00 6.3 ± 0.32 8.0 ± 0.25 * 8.4 ± 0.15 * 8.9 ± 0.25 * 8.8 ± 0.23 * 5.6 ± 0.15 7.9 ± 0.49 * 8.2 ± 0.25 * 8.7 ± 0.42 * 8.9 ± 0.33 * 8.9 ± 0.14 * 5.4 ± 0.07 8.4 ± 0.12 * 9.4 ± 0.00 * 9.4 ± 0.10 * 9.3 ± 0.10 * 10.0 ± 0.06 * 5.3 ± 0.07
5.0 ± 0.00 6.8 ± 0.10 8.2 ± 0.00 * 8.5 ± 0.06 * 9.0 ± 0.00 * 9.0 ± 0.17 * 5.4 ± 0.00 8.0 ± 0.21 * 8.3 ± 0.10 * 8.8 ± 0.21 * 9.1 ± 0.15 * 9.5 ± 0.14 * 5.0 ± 0.21 8.4 ± 0.23 * 8.5 ± 0.21 * 9.6 ± 0.36 * 9.4 ± 0.12 * 10.0 ± 0.15 * 5.1 ± 0.14
4.9 ± 0.35 5.4 ± 0.06 8.0 ± 0.06 * 9.6 ± 0.12 * 9.0 ± 0.20 * 9.1 ± 0.15 * 5.3 ± 0.22 8.2 ± 0.28 * 9.2 ± 0.05 * 9.5 ± 0.71 * 8.9 ± 0.14 * 9.0 ± 0.07 * 4.8 ± 0.42 8.1 ± 0.25 * 9.3 ± 0.00 * 9.2 ± 0.15 * 9.6 ± 0.10 * 9.4 ± 0.06 * 5.2 ± 0.07
5.3 ± 0.00 5.7 ± 0.32 7.8 ± 0.21 * 8.3 ± 0.06 * 9.4 ± 0.06 * 9.2 ± 0.17 * 5.2 ± 0.10 7.8 ± 0.00 * 8.7 ± 0.31 * 9.1 ± 0.21 * 9.4 ± 0.10 * 9.1 ± 0.07 * 5.1 ± 0.07 8.4 ± 0.10 * 9.0 ± 0.12 * 9.4 ± 0.12 * 9.8 ± 0.15 * 9.7 ± 0.25 * 5.0 ± 0.10
4.9 ± 0.42 6.4 ± 0.35 7.9 ± 0.40 * 8.5 ± 0.26 * 9.4 ± 0.06 * 9.2 ± 0.15 * 5.0 ± 0.33 7.8 ± 0.00 * 8.6 ± 0.24 * 9.1 ± 0.21 * 9.5 ± 0.06 * 9.2 ± 0.07 * 4.8 ± 0.00 8.2 ± 0.20 * 8.8 ± 0.12 * 9.1 ± 0.06 * 9.9 ± 0.06 * 9.6 ± 0.10 * 4.9 ± 0.11
20 V
30 V
Passive diffusion
The pH increased in a duty cycle-dependent manner at 10, 20 and 30 V. The pH declined in the case of passive diffusion and with the application of a 50% duty cycle, at each of the 3 voltages. mean ± S.D. (n = 5). *p b 0.05 versus 50% duty cycle.
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is the electroosmotic flux, which is the convective movement of a solvent through a charged pore in response to the preferential passage of counter-ions when an electric field is applied. Jp and Jer, that is the movement of ions in response to concentration gradient and an electric field, is described by the Nernst-Planck equation, Jp + Jer = −Di
dCi zF d/ Ci + dx RT dx
ð6Þ
where Di is the diffusion coefficient in the membrane, Ci is the donor concentration of the ionic species i, φ is the electrical potential, z is the charge of the permeant, F is the Faraday constant, R is the universal gas constant, and T is the absolute temperature. Jeo that is the additional component of flux is associated with the solvent itself being in motion. In the electroosmotic flux, the velocity of the ionic species i is equal to that of the solvent, that is expressed as follows. vi = v
ð7Þ
where vi and v is the average velocity of the ionic species i near the membrane and that of the solvent respectively. Therefore Jeo is given by Jeo = v Ci
ð8Þ
where Ci is the average concentration of the ionic species i near the membrane. In the present study, the lidocaine transport efficiency increased with increasing voltage and duty cycle. This result suggests that the width of the square wave, as the DC component, plays an important role in the generation of electrorepulsion and the periodic polarity alteration with the introduction of the square wave is involved in electroosmosis. Electroosmosis is a phenomenon which an electrically driven flow of ions across a membrane with a net charge can induce the coupled flow of solvent [13]. A bulk motion of the solvent itself produced by electroosmosis carries ions or neutral species across a membrane, within the solvent stream. According to our previous studies [8–10], electrorepulsion and electroosmosis are closely involved in the transport of lidocaine with the application of AC. Yan and Li et al. accomplished successful transport of a charged permeant, tetraethyl ammonium, and a neutral permeant, arabinose, across a synthetic membrane by application of a square-wave AC iontophoresis. Their report suggested that both electrorepulsion and electroosmosis play essential roles in the transport of substances in AC fields, although the electroosmotic enhancement was smaller than the electrorepulsive effect [4]. Electroosmosis frequently occurs in DC fields, but can also be observed in AC fields. In the present study, not only electrorepulsion but also electroosmosis were probably involved in the enhancement of lidocaine transport caused by AC delivery with duty cycle. However, the relative contributions of electrorepulsion and electroosmosis to the total iontophoretic flux have been proven difficult to quantify, due to the difficulty of designing appropriate experiment. The relative importance of electrorepulsion and electroosmosis depends on the physiochemical and electrical characteristics of the membrane and of the permeant. 4.3. pH changes and the electrode It has been reported that various physical and chemical properties of the donor substances, such as the concentration of the substance, pH, dissociation state of ions, molecular size, molecular weight, charge state, lipid solubility, water solubility and chemical stability, influence the
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transport efficiency [13]. Previous reports have described that both the pH environment of the skin or solution and the degree of drug ionization are involved in iontophoretic drug transport [16,17]. In the present study, the pH of the solution in the receptor chamber showed duty cycle-dependence under the application of 10, 20 and 30 V. Voltagedependence of the pH was also observed at duty cycles of 60%, 70%, 80%, 90% and 95%. These findings indicate that the water in the chamber was electrolyzed, resulting in the production of OH− ions in the receptor chamber. The reaction rate of electrolysis increased depending on the voltage and the duty cycle. The production of ions may reduce the flux of similarly charged solute ions. On the other hand, H+ ions produced by electrolysis would compete with the positively charged lidocaine ions under such circumstances, resulting in decrease of the iontophoretic drug flux, because they carry a fraction of the total current. However, the pH decreased with time with the application of a duty cycle of 50% at each voltage and during passive diffusion. The pH decrease associated with passive diffusion indicates that the dissociated lidocaine hydrogen ion (C14H22N2O∙H+) spontaneously permeated from the donor chamber to the receptor chamber without water electrolysis, resulting in the accumulation of H+ ions. The pH decrease observed with a 50% duty cycle was due to accumulation of dissociated lidocaine hydrogen ions transported by the application of an electric field, and suggests a lower degree of water electrolysis. The electrode materials affect the pH of the solution. In the present study, platinum electrodes were used in order to prevent the generation of competing ions. The electrodes used for iontophoresis are classified roughly into two categories, namely, the inert type and the reversible type. The inert-type electrodes, such as platinum and aluminum, do not take part in electrochemical reactions, but cause electrolysis of water, resulting in the production of H+ at the anode and OH− at the cathode. On the other hand, the reversible- type electrodes, such as silver/silver chloride (Ag|AgCl) electrodes, are resistant to pH changes, because of the oxidation reaction of the electrodes expressed as follows. Ag þ Cl− ¼ AgCl þ e−
ð9Þ
This oxidation reaction of the electrode produces insoluble silver chloride, which precipitates on the anode surface, resulting in the absence of net migration of silver ions into the donor chamber. However, the reversible-type electrodes generate competing ions resulting in a decrease of the transport efficiency [13]. 5. Conclusion Our experimental system was successfully used to evaluate the effect of the waveform component of high-frequency AC voltage on lidocaine transport across a cellophane membrane. The results of this study indicated that lidocaine was transported efficiently through a cellophane membrane with the use of square-wave AC iontophoresis with a duty cycle, in a voltage-, time- and duty cycle-dependent manner. With the application of 30−V, 1 kHz AC voltage with a 95% duty cycle for 60 min, the lidocaine concentrations were about twofold higher than that observed with the application of a 50% duty cycle and about threefold higher than that observed following passive diffusion, however, the pH of the solution in the receptor chamber increased markedly because of water electrolysis. When an AC voltage of 10 V was applied with a 70% duty cycle or of 20 V was applied with a 60% duty cycle, lidocaine was transported more efficiently as compared with that observed with a 50% duty cycle, with also a smaller change of the pH of the solution in the receptor chamber. Consequently, it is suggested that application of 10 V with a 70% duty cycle or 20 V with a 60% duty cycle is optimal for lidocaine transport in clinical situations, because of the lesser degree of water electrolysis as compared with that observed with the application of 30 V with a 95% duty cycle.
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Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research No.14207088 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] R. Rai, C.R. Srinivas, Iontophoresis in dermatology, Indian J. Dermatol. Venereol. Leprol. 71 (2005) 236–241. [2] E.R. Viscusi, L. Reynolds, S. Tait, T. Melson, L.E. Atkinson, An iontophoretic fentanyl patient-activated avalgegic delivery system for postoperative pain: a double-blind, placebo-controlled trial, Anesth. Analg. 102 (2006) 188–194. [3] G.L. Li, T.J. Van Steeg, H. Putter, J. Van Der Spek, S. Pavel, M. Danhof, J.A. Bouwstra, Cutaneous side-effects of transdermal iontophoresis with and without surfactant pretreatment: a single-blinded, randomized controlled trial, Br. J. Dermatol. 153 (2005) 404–412. [4] G. Yan, S.K. Li, K.D. Peck, H. Zhu, W.I. Higuchi, Quantitative study of electrophoretic and electroosmotic enhancement during alternating current iontophoresis across synthetic membranes, J. Pharm. Sci. 93 (2004) 2895–2908. [5] J.P. Howard, T.R. Drake, D.L. Kellogg Jr., Effects of alternating current iontophoresis on drug delivery, Arch. Phys. Med. Rehabil. 76 (1995) 463–466. [6] G. Yan, K.D. Peck, H. Zhu, W.I. Higuchi, S.K. Li, Effects of electrophoresis and electroosmosis during alternating current iontophoresis across human epidermal membrane, J. Pharm. Sci. 94 (2005) 547–558.
[7] T. Shibaji, Y. Yasuhara, N. Oda, M. Umino, A mechanism of the high frequency AC iontophoresis, J. Control. Release 73 (2001) 37–47. [8] H. Izumikawa, Lidocaine transportation through a cellophane membrane by wide range AC frequencies, J. Stom. Soc. 72 (2005) 183–189. [9] T. Kinoshita, T. Shibaji, M. Umino, Transdermal delivery of lidocaine in vitro by alternating current, J. Med. Dent. Sci. 50 (2003) 71–77. [10] H. Haga, T. Shibaji, M. Umino, Lidocaine transport through living rat skin using alternating current, Med. Biol. Eng. Comput. 43 (2005) 622–629. [11] S. Reinauer, A. Neusser, G. Schauf, E. Hölzle, Iontophoresis with alternating current and direct current offset (AC/DC iontophoresis): a new approach for the treatment of hyperhidrosis, Br. J. Derm. 153 (1993) 166–169. [12] K. Okabe, H. Yamaguchi, Y. Kawai, New iontophoretic transdermal administration of the beta-blocker metoprolol, J. Control. Release 4 (1986) 79–85. [13] P. Singh, H.I. Maibach, Iontophoresis in drug delivery: basic principles and applications, Crit. Rev. Ther. Drug Carr. Syst. 11 (1994) 161–213. [14] Muh-Hwan Su, V. Srinvasan, Abdel-Halim Ghanem, W.I. Higuchi, Quantitative in vivo iontophoretic studies, J. Pharm. Sci. 83 (1994) 12–17. [15] P. Santi, P.L. Catellanin, G. Massimo, G. Zanardi, P. Colombo, Iontophoretic transport of verapamil and melatonin I. Cellophane membrane as a barrier, Int. J. Pharm. 92 (1993) 23–28. [16] V. Merino, A. López, Y.N. Kalia, R.H. Guy, Electrorepulsion versus electroosmosis: effect of pH on the iontophoretic flux of 5-fluorouracil, Pharm. Res. 16 (1999) 758–761. [17] N. Vinod, P. Ramesh, Physicochemical considerations in the iontophoretic delivery of a small peptide: in vitro studies using arginine vasopressin as a model peptide, Pharm. Res. 48 (2003) 175–182.
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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m
Lidocaine transport through a cellophane membrane by alternating current iontophoresis with a duty cycle Shizuka Hayashi a,⁎, Saori Ogami a, Takao Shibaji b, Masahiro Umino a a Section of Anesthesiology and Clinical Physiology, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Section of Orofacial Pain Management, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
a r t i c l e
i n f o
Article history: Received 28 March 2008 Received in revised form 12 November 2008 Accepted 18 November 2008 Available online 30 November 2008 Keywords: Iontophoresis Alternating current Lidocaine Duty Cycle Cellophane membrane
a b s t r a c t The purpose of this study was to determine whether lidocaine can be efficiently transported across a cellophane membrane using a square-wave alternating current (AC) with an adjusted duty cycle. Three voltages at 1 kHz with 6 duty cycles were applied for 60 min to the diffusion cells on both sides of the cellophane membrane. The donor chamber was filled with 1% lidocaine hydrochloride solution. The transport of lidocaine was enhanced in a voltage-, and duty cycle-dependent manner. These findings indicate that voltage and the direct current (DC) component of the square-wave AC play important roles in generating the driving force necessary for lidocaine delivery. Additionally, the periodic polarity alteration could reduce the electrode polarization. The higher voltages and duty cycles induced a pH change. The practical electrical conditions which are preferable for clinical application were 10 V with a 70% duty cycle or 20 V with a 60% duty cycle. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Iontophoresis is a delivery method by which the movement of ionic compounds or non-charged drug molecules is affected by the application of an external electric field. Iontophoresis enhances the transport of ionic substances across membranes or the skin via three principal mechanisms: electrorepulsion, electroosmosis and electroporation. Among these, electrorepulsion is considered to play the most crucial role in the transport of ionic drugs. Transdermal drug delivery using iontophoresis has a number of potential advantages, e.g., drug degradation due to passage through the stomach or intestine or the first-pass through the liver is avoided, it is user-friendly, and it is a painless method. Transdermal iontophoresis using a direct current (DC) has been used for both local and systemic drug delivery. Applications include local delivery of anesthetics, steroids and retinoids to treat scarring caused by acne, for the relief of palmar and plantar hyperhidrosis [1], and iontophoresis of fentanyl is also used for the management of postoperative pain or cancer pain [2]. However, some adverse effects, including electrical burns or erythema as a result of electrode polarization during electrolysis, have been reported in clinical situations [3]. This negative side effect limits the application time of DC iontophoresis to less than 15 min at current densities as low as 1 mA/cm2 [4]. Furthermore, the transport efficiency is also reduced with increasing duration of the electrical application. The lowered transport efficiency is caused by a ⁎ Corresponding author. Tel.: +81 3 5803 5549; fax: +81 3 5803 0206. E-mail address: [email protected] (S. Hayashi). 1567-5394/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2008.11.007
voltage drop in the solution resulting from the formation of an electric double layer on the electrode surface (a phenomenon known as ‘electrode polarisation’), because of the accumulation of ionized substances with a different charge from that of the electrode polarity. To resolve this problem, alternating current (AC) iontophoresis has also been studied. Howard et al. reported that the AC-iontophoretic transport of hydroxocobalamin through human skin was more efficient than passive transport [5]. Yan and Peck et al. applied square-wave AC iontophoresis across a synthetic membrane and confirmed that flux of tetraethyl ammonium and arabinose were enhanced by increasing the voltage and decreasing the frequency of the AC [6]. We have also studied ion transport using AC. Shibaji et al. revealed that physiological saline could be transported through a cellophane membrane using a sinusoidal AC voltage at 1 kHz [7]. Izumikawa reported transport of lidocaine through a cellophane membrane using AC, and the transport efficiency was markedly enhanced at 1 kHz [8]. Kinoshita et al. found that lidocaine could be transported through excised rat skin in vitro using a sinusoidal AC voltage [9]. We succeeded in transport of lidocaine through living rat skin using AC in a voltage- and time-dependent manner [10]. Furthermore, Yan and Peck et al. reported that AC with a DC offset voltage enhances the transport of both neutral and ionic permeants [6]. On the other hand, Reinauer et al. found no effect of the application of sawtooth wave AC iontophoresis for the treatment of palmar hyperhidrosis [11]. These reports suggest that AC with a DC component may be useful for the transport of ionic compounds. The degree of electrode polarization on the electrode or skin surface in the case of application of AC is less than that in the case of DC,
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because of the periodic polarity alteration. Accordingly, the decrease of the transport efficiency of ionic compounds with increasing time is alleviated. However, the transport efficiency of ionic compounds by electrorepulsion in AC is inferior to that by DC, because AC has no direct current component. If AC is combined with a DC component, electrorepulsion due to the DC component can be expected despite the periodic polarity alteration. Therefore, we applied a bipolar square wave with a duty cycle for lidocaine transport across the cellophane membrane. The purpose of this study was to determine whether lidocaine can be efficiently transported across a cellophane membrane using a square-wave AC with an adjusted duty cycle, and to find the optimal duty cycle and voltage for such lidocaine transport. 2. Materials and methods 2.1. Materials Lidocaine hydrochloride (C14H22N2O∙HCl, FW: 270.8, H2O content: 1 mol/mol) was purchased from Sigma-Aldrich Co. Ltd. (St. Louis, USA). One percent lidocaine hydrochloride was prepared using distilled and deionized water (resistivity N18 MΩ q cm). The pH of the 1% lidocaine hydrochloride solution was 4.6. The solution was degassed using an ultrasonic cleaner for 30 min immediately before injection into the chamber. 2.2. Membrane The cellophane membrane (Futamura Chemical Co., Ltd., Nagoya, Japan) was about 36 μm thick, with a pore size of about 2–3 nm. This pore size was about twofold larger than the size of the lidocaine molecules. 2.3. Experimental cell and transport system A cylindrical acryl drug delivery cell consisting of two chambers was originally constructed. Platinum plate electrodes with a diameter of 20 mm and thickness of 0.2 mm were installed at opposite ends of the two components of the cell. The length of each component was 10 mm (Fig. 1). The cellophane membrane was sandwiched between the two components (one of which was the donor chamber
and the other, the receptor chamber). The available diffusion area was about 3.1 cm2 and the capacity of the each chamber was about 3.0 ml. The donor chamber was filled with 1% lidocaine hydrochloride solution (3.0 ml, pH 4.6) and the receptor chamber with distilled water (3.0 ml). The acryl cells were set in the water bath, and the temperature of the cell was controlled so as to not allow it to exceed 36.5 °C. A thermocouple microprobe (BAT-12, Physitemp, NJ, USA) was inserted at the center of the donor chamber to monitor the temperature of the solution contained in it. The solution in the two cell chambers was not stirred, because the bulk flow caused by stirring and the rotator-induced magnetic field can affect lidocaine transport. The aqueous boundary layer due to the absence of stirring in the cell was negligible. Constant-voltage AC was continuously applied between the parallel platinum electrodes for 60 min using a function/arbitrary waveform generator (Agilent 33250A, Agilent Technologies, Colorado, USA) and a high speed power amplifier (4025, NF Electric Instruments, Kanagawa, Japan), and the waveform and voltage of output were monitored with a digitizing oscilloscope (HP54503A, Hewlett Packard, Tokyo, Japan) throughout the duration of the experiment. 2.4. Determination of the lidocaine concentration and pH Twenty μl of solution was sampled every 10 min after the start of the AC application for 60 min, with a micropipette (20–200 μl, Nichiryo, Tokyo, Japan) placed at the center of the receptor chamber. The concentration of lidocaine in the receptor chamber was determined using a spectrophotometer (U-3310; absorbance range: −2∼4 Abs; precision: ±0.002 Abs, HITACHI, Tokyo, Japan) at room temperature. Before the analysis, the samples were diluted 40 times with distilled and deionized water. The absorbance values of the samples were measured at a wavelength of 262 nm and optical path of 10 mm. The concentration of lidocaine was quantified using a calibration curve. The detection limit was 11.6 μg/ml. The pH of the samples was measured with an FET (Field Effect Transistor) pH meter (pH BOY-P2, Shindengen Electric MFG Co., Tokyo, Japan). The efficiency of iontophoresis was defined as the slope of the timecourse of increase in the transport of lidocaine from the donor chamber to the receptor chamber during the experiment. The efficiency of
Fig. 1. The setup of the experimental system. The experimental system consists of two chambers, a thermocouple microprobe, a water bath, a function generator and a high speed power amplifier.
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2.6. Statistical analysis All values are presented as mean ± S.D. (n = 5). A two-way ANOVA test was used to analyze the time- and duty cycle-dependences of the transport efficiency of lidocaine. In addition, the Tukey test was used to analyze the duty cycle- and voltage-dependences of the transport
Fig. 2. Diagram of a square-wave AC with a 100a% duty cycle (a = 0.5, 0.6, 0.7, 0.8, 0.9, 0.95). T is the period of the square wave. A 100a% duty cycle represents the ratio of the positive cycle to the full cycle. The ratio of the positive cycle was adjusted between 50%, 60%, 70%, 80%, 90% and 95%.
iontophoresis was calculated by applying the least-square method, expressed by Eq. (1) as follows: 6
2
y = ax + b; r 2 = ∑ ½yi −ðaxi + bÞ ; i=1
Ar 2 Ar 2 = 0; =0 Aa Ab
ð1Þ
where xi (i = 1, …, 6) represents the time [min] (x1 = 10, x2 = 20, …, x6 = 60), yi is the concentration of lidocaine in the receptor chamber [μmol/cm3], a is the slope of the time-course of increase in the transport of lidocaine [μmol/cm3/min], b is a constant, and r is the correlation coefficient. The variable a is defined as the efficiency of iontophoresis, which is calculated by the gradient of the approximate linear equation fitting the time-course curves for ion concentration in the receptor chamber during AC iontophoresis. In addition, the flux [μmol/h/cm2] of lidocaine through cellophane membrane was calculated from the cumulative amount of lidocaine transported to the receptor chamber over a period of 60 min. 2.5. Experimental design To examine the relationship between the efficiency of lidocaine transport and the duty cycle, a bipolar square wave with 6 different duty cycles, that is, 50%, 60%, 70%, 80%, 90% and 95%, was continuously applied at 1 kHz for 60 min to the drug delivery cell, with the donor chamber containing 1% lidocaine solution, under 3 different constant voltages of 10, 20 and 30 V, using a function/arbitrary waveform generator (Agilent 33250A, Agilent Technologies, Colorado, USA) and a high speed power amplifier (4025, NF Electric Instruments, Kanagawa, Japan) (Fig. 2). The lidocaine concentration in the receptor chamber was determined by spectrophotometry (U-3310; absorbance range: −2∼4 Abs; precision: ±0.002 Abs, HITACHI, Tokyo, Japan) at 10, 20, 30, 40, 50 and 60 min after the start of the current application. The pH of the solution in the receptor chamber was measured with an FET pH meter (pH BOY-P2, Shindengen Electric MFG Co., Tokyo, Japan) at 10, 20, 30, 40, 50 and 60 min after the start of the current application for each duty cycle and voltage condition.
Fig. 3. Changes of the lidocaine concentrations for applied a square-wave AC with 6 different duty cycles under (a) 10 V, (b) 20 V and (c) 30 V at 1 kHz: (○) passive, (●) duty cycle 50%, (□) 60%, (■) 70%, (△) 80%, (▲) 90%, (▽) 95%. The error bars represent standard deviation of the mean. The lidocaine concentrations increased significantly in a time-dependent and approximately linear manner for every duty cycle.
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efficiency of lidocaine and the relationship between the duty cycle and pH. Statistical significance was set at p b 0.05. 3. Results
On the other hand, the pH of the solution in the donor chamber declined in a duty cycle-dependent manner at 10, 20 and 30 V. With the 95% duty cycle at 10 V and the 80%, 90%, and 95% duty cycles at 20 V and 30 V that were applied for 60 min, the pH of the donor chamber was about 1.8.
3.1. Lidocaine concentration and duty cycles 4. Discussion Fig. 3a–c shows the changes of the lidocaine concentrations in the receptor chamber induced by the application of a square-wave AC with 6 different duty cycles, that is, 50%, 60%, 70%, 80%, 90% and 95%, under 10, 20 or 30 V at 1 kHz. The lidocaine concentrations in the receptor chamber increased significantly in a timedependent and approximately linear manner for every duty cycle under 10 V (F = 5.49, p b 0.001; Fig. 3a), 20 V (F = 5.33, p b 0.001; Fig. 3b) and 30 V (F = 8.75, p b 0.001; Fig. 3c). The lidocaine concentration obtained with application of the 95% duty cycle at 30 V was about twofold higher than that obtained with application of the 50% duty cycle, and about threefold higher than that observed following passive diffusion, at 60 min after the start of the current application (Fig. 3c). Fig. 4a–c shows the lidocaine transport efficiency obtained for the 6 different duty cycles under 10 V (Fig. 4a), 20 V (Fig. 4b) and 30 V (Fig. 4c) at 1 kHz. The lidocaine transport efficiency increased in a linear manner as the duty cycle increased, at each of the 3 voltages used. At 10 and 20 V, the lidocaine transport efficiencies with the application of 70%, 80%, 90% and 95% duty cycles were significantly higher as compared with the efficiency obtained with a 50% duty cycle (p b 0.05; Fig. 4a–b). At 30 V, the lidocaine transport efficiency with the application of duty cycles of 60%, 70%, 80%, 90% and 95% were significantly higher compared with that observed with a 50% duty cycle (p b 0.001; Fig. 4c). 3.2. Lidocaine transport efficiency and voltages At the duty cycle of 50%, the lidocaine transport efficiency increased with increase of voltage, however, there were no significant differences in the lidocaine transport efficiency among the three voltages examined. Although the lidocaine transport efficiency at 30 V was significantly higher than that at 10 and 20 V (p b 0.05) for the duty cycles of 60%, 70%, 80% and 90%, no significant differences in the lidocaine transport efficiency were observed between 10 and 20 V for any width of the duty cycles. At the duty cycle of 95%, the lidocaine transport efficiency at 30 V was significantly higher than that at 20 V, and that at 20 V was significantly higher than the efficiency at 10 V (p b 0.05). Fig. 5 shows the relationship between the voltage and the flux of lidocaine. The lidocaine flux depended on the applied voltage. At the duty cycle of 95%, the average flux under the application of 30 V for 60 min was about 1.3-fold higher than that obtained with application of 10 V, and about threefold higher than that observed following passive diffusion.
4.1. Square-wave AC and lidocaine transport efficiency The present study revealed that application of a square-wave AC with a duty cycle, which has both components of direct and alternating currents, was effective for accomplishing successful transport of lidocaine in a time-, voltage-, and duty cycle-dependent manner. These findings indicate that the voltage and the DC component of the square-wave AC play important roles in generating the driving force necessary for lidocaine delivery. Additionally, the periodic polarity alteration could reduce the electrode polarization, which disturbs the transport of lidocaine ions, resulting in enhancement of the lidocaine transport efficiency. Application of an electric field causes a polarization of the skin or the electrode surface oriented in the direction opposite to the applied field. Marked electrode polarization occurs with the application of DC alone. The effective current diminishes with increase of the DC application time, because electrode polarization of the skin and the electrode surface act as a capacitor in an electric circuit. Polarization lowers the transport efficiency of substances. Until now, various waveforms have been used to avoid polarization of the electrode surface and skin in iontophoresis. Iontophoresis using AC in combination with DC has also been attempted for the treatment of palmoplantar hyperhidrosis, and was shown to be associated with reduced side effects [11]. Pulsed DC with duty cycles ranging from 20% to 80% has been studied both at the basic and clinical levels [11–13]. To decrease the electrode polarization, we have previously attempted application of a sine-wave AC at various frequencies and voltages for lidocaine transport, both in vitro and in vivo [8–10]. However, the driving force with the use of a sine-wave AC is inferior to that with the application of DC, because of the periodic polarity alteration. Therefore, a square wave with polarity alteration (square-wave AC) was employed in the present study. In the iontophoretic study, artificial membranes, animal or human skins have been used as a partition placed between donor and receptor cells. In the present study, cellophane membrane was employed because it has not been investigated whether ionized substances can be transported through an artificial partition by a square wave with polarity alteration. The AC component with the periodic polarity alteration generates less electrode polarization and the square wave generates the driving force for the drug. The average voltage Uave for one period of the square wave can be expressed as 1 T ∫ U ðt Þdt T 0
ð2Þ
3.3. Duty cycle and pH change
Uave =
Table 1 shows the pH of the solution in the receptor chamber for the application of a square-wave AC with 6 different duty cycles, that is, 50%, 60%, 70%, 80%, 90% and 95% under 10, 20 and 30 V, at 1 kHz. The pH of the solution in the receptor chamber increased in a duty cycle-dependent manner at 10, 20 and 30 V. The pH declined in the case of passive diffusion and with the application of a 50% duty cycle, at 10, 20 and 30 V. There were no significant differences in the pH between duty cycles of 50% and 60% at 10 V. The pH following application of duty cycles of 70%, 80%, 90% and 95% was significantly higher than that observed with a 50% duty cycle at 10 V (p b 0.05). The pH following the application of duty cycles of 60%, 70%, 80%, 90% and 95% was significantly higher than that observed with the application of a 50% duty cycle at 20 V and 30 V (p b 0.05).
where t is the time, U(t) is the voltage between the electrodes at t, and T is the period of the square wave. When a constant AC voltage A with a 100a (%) duty cycle was applied, the voltage between the electrodes is A from 0 (s) to aT (s) after the start of the current application, and –A from aT (s) to T (s) (Fig. 2). Therefore, Eq. (2) can be reduced to Uave =
o 1 n aT T ∫ 0 Adt + ∫aT ð−AÞdt T = ð2a−1ÞA
ð3Þ
where a is the duty cycle (a = 0.5, 0.6, 0.7, 0.8, 0.9, 0.95), and A is the amplitude of the square-wave AC. From Eq. (3), it can be seen that Uave
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Fig. 4. The change of the lidocaine transport efficiency as a function of the duty cycle of square-wave AC under (a) 10 V, (b) 20 V and (c) 30 V at 1 kHz. The efficiency of lidocaine transport increased in a linear manner as the duty cycle increased, at each of the 3 voltages.
remains constant with respect to time when voltage and duty cycle are constant. And Uave can be regarded as the DC component and effective for penetration of drug. From Eq. (3), it is seen that Uave is a linear function of the duty cycle when a constant AC voltage is applied, and
when the duty cycle is constant, the Uave is proportional to the voltage between the electrodes. In the present study, the lidocaine transport efficiency showed voltage dependence at all the duty cycles applied. This finding indicates that
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Fig. 5. The change of the lidocaine flux as a function of the applied voltage when a 95% duty cycle at 1 kHz was applied. The flux of lidocaine increased in a voltage-dependent manner.
the voltage is closely related to the driving force. Our previous in-vivo and in-vitro studies using the sine-wave AC also revealed voltage dependence of lidocaine transport [8,9]. The frequency of the current and the degree of ionization of a substance are also known to influence the transport efficiency. In our study, the frequency of 1 kHz was employed, because our previous study results showed that lidocaine can be effectively transported at this frequency [9]. A 1% lidocaine solution was used in the present study. Lidocaine, with a molecular weight of 234.3, is composed of a lipophilic part (a benzene ring), a hydrophilic part (a tertiary amine), and an intermediate carboxy linkage. For commercial use, lidocaine is used as its hydrochloride salt. Lidocaine hydrochloride (salt) is quite soluble in water. In aqueous solution, the lidocaine hydrochloride molecule dissociates and ionizes to yield a quaternary amine cation plus an acid anion. In the present study, the pH of the lidocaine solution in the donor chamber ranged from 4.6 to 1.8. Since the pKa of lidocaine is 7.86, the lidocaine molecules were almost completely dissociated and ionized in the aforementioned pH range. Lidocaine molecules dissociated into cation were transported to the receptor chamber by the square-wave AC.
Drug ion transport is a function of the total current and the fluxes of other ions present. The transport rates of other species are also dependent upon the total current. The donor chamber used in the present study contained positively charged lidocaine ions and negatively charged chloride ions. The ion current was produced by movement of the lidocaine ions as well as that of the other ions in the donor chamber with the AC application [14]. The transport rate of a drug depends on its transference number (the fraction of the current carried by the drug ion) and the total current driven, which in turn, is related to the voltage drop across the rate-limiting resistance to transport of the drug [14]. Although the lidocaine transport efficiency was the most effective with the use of the 95% duty cycle at 30 V in the present study, the pH of the solution in the receptor chamber was markedly increased under this condition because of water electrolysis. Application of a duty cycle of 70% at 10 V or 60% at 20 V produced effective transport as compared with that observed with the use of a 50% duty cycle, with a smaller change of the pH of the solution in the receptor chamber. These results suggest that application of a duty cycle of 70% at 10 V or 60% at 20 V is preferable for clinical application, because of the less marked occurrence of water electrolysis as compared with that observed with a 95% duty cycle at 30 V. 4.2. Transport mechanism The present study demonstrated that the transport efficiency of lidocaine depended on the duty cycle at each of the AC voltages examined. This finding shows that the width of the DC component in the square wave was closely associated with the driving force of lidocaine. The mole flux of substance i, which is named Ji, is defined as Ji = ðdni =dt Þ=A
ð4Þ
where ni is the number of substance i and A the transporting area. Although the transport mechanism of substances driven by the application of AC with a duty cycle has not yet been completely elucidated, the following Eq. (5) may explain the substance transport mechanism with the application of an external electric field [13,15]. ð5Þ
Ji = Jp + Jer + Jeo
where Jp is the passive flux, Jer is the electrorepulsive contribution, in which the ion is repelled from an electrode of the same charge, and Jeo
Table 1 The pH changes of the solution in the receptor chamber Applied voltage
Duty cycle
t (min) 0
10
20
30
40
50
60
10 V
50% 60% 70% 80% 90% 95% 50% 60% 70% 80% 90% 95% 50% 60% 70% 80% 90% 95%
6.4 ± 0.00 6.5 ± 0.02 6.4 ± 0.02 6.5 ± 0.01 6.5 ± 0.06 6.4 ± 0.02 6.4 ± 0.00 6.4 ± 0.07 6.4 ± 0.01 6.4 ± 0.04 6.5 ± 0.06 6.5 ± 0.07 6.4 ± 0.21 6.3 ± 0.25 6.4 ± 0.17 6.4 ± 0.10 6.4 ± 0.12 6.4 ± 0.06 6.1 ± 0.00
5.4 ± 0.28 6.4 ± 0.15 7.8 ± 0.42 * 7.8 ± 0.26 * 8.4 ± 0.56 * 8.8 ± 0.15 * 5.7 ± 0.00 7.6 ± 1.20 * 8.1 ± 0.24 * 8.6 ± 0.49 * 8.8 ± 0.01 * 9.4 ± 0.00 * 5.7 ± 0.00 8.2 ± 0.15 * 8.9 ± 0.10 * 8.8 ± 0.15 * 9.5 ± 0.15 * 9.3 ± 0.17 * 6.0 ± 0.06
5.1 ± 0.00 6.3 ± 0.32 8.0 ± 0.25 * 8.4 ± 0.15 * 8.9 ± 0.25 * 8.8 ± 0.23 * 5.6 ± 0.15 7.9 ± 0.49 * 8.2 ± 0.25 * 8.7 ± 0.42 * 8.9 ± 0.33 * 8.9 ± 0.14 * 5.4 ± 0.07 8.4 ± 0.12 * 9.4 ± 0.00 * 9.4 ± 0.10 * 9.3 ± 0.10 * 10.0 ± 0.06 * 5.3 ± 0.07
5.0 ± 0.00 6.8 ± 0.10 8.2 ± 0.00 * 8.5 ± 0.06 * 9.0 ± 0.00 * 9.0 ± 0.17 * 5.4 ± 0.00 8.0 ± 0.21 * 8.3 ± 0.10 * 8.8 ± 0.21 * 9.1 ± 0.15 * 9.5 ± 0.14 * 5.0 ± 0.21 8.4 ± 0.23 * 8.5 ± 0.21 * 9.6 ± 0.36 * 9.4 ± 0.12 * 10.0 ± 0.15 * 5.1 ± 0.14
4.9 ± 0.35 5.4 ± 0.06 8.0 ± 0.06 * 9.6 ± 0.12 * 9.0 ± 0.20 * 9.1 ± 0.15 * 5.3 ± 0.22 8.2 ± 0.28 * 9.2 ± 0.05 * 9.5 ± 0.71 * 8.9 ± 0.14 * 9.0 ± 0.07 * 4.8 ± 0.42 8.1 ± 0.25 * 9.3 ± 0.00 * 9.2 ± 0.15 * 9.6 ± 0.10 * 9.4 ± 0.06 * 5.2 ± 0.07
5.3 ± 0.00 5.7 ± 0.32 7.8 ± 0.21 * 8.3 ± 0.06 * 9.4 ± 0.06 * 9.2 ± 0.17 * 5.2 ± 0.10 7.8 ± 0.00 * 8.7 ± 0.31 * 9.1 ± 0.21 * 9.4 ± 0.10 * 9.1 ± 0.07 * 5.1 ± 0.07 8.4 ± 0.10 * 9.0 ± 0.12 * 9.4 ± 0.12 * 9.8 ± 0.15 * 9.7 ± 0.25 * 5.0 ± 0.10
4.9 ± 0.42 6.4 ± 0.35 7.9 ± 0.40 * 8.5 ± 0.26 * 9.4 ± 0.06 * 9.2 ± 0.15 * 5.0 ± 0.33 7.8 ± 0.00 * 8.6 ± 0.24 * 9.1 ± 0.21 * 9.5 ± 0.06 * 9.2 ± 0.07 * 4.8 ± 0.00 8.2 ± 0.20 * 8.8 ± 0.12 * 9.1 ± 0.06 * 9.9 ± 0.06 * 9.6 ± 0.10 * 4.9 ± 0.11
20 V
30 V
Passive diffusion
The pH increased in a duty cycle-dependent manner at 10, 20 and 30 V. The pH declined in the case of passive diffusion and with the application of a 50% duty cycle, at each of the 3 voltages. mean ± S.D. (n = 5). *p b 0.05 versus 50% duty cycle.
S. Hayashi et al. / Bioelectrochemistry 74 (2009) 315–322
is the electroosmotic flux, which is the convective movement of a solvent through a charged pore in response to the preferential passage of counter-ions when an electric field is applied. Jp and Jer, that is the movement of ions in response to concentration gradient and an electric field, is described by the Nernst-Planck equation, Jp + Jer = −Di
dCi zF d/ Ci + dx RT dx
ð6Þ
where Di is the diffusion coefficient in the membrane, Ci is the donor concentration of the ionic species i, φ is the electrical potential, z is the charge of the permeant, F is the Faraday constant, R is the universal gas constant, and T is the absolute temperature. Jeo that is the additional component of flux is associated with the solvent itself being in motion. In the electroosmotic flux, the velocity of the ionic species i is equal to that of the solvent, that is expressed as follows. vi = v
ð7Þ
where vi and v is the average velocity of the ionic species i near the membrane and that of the solvent respectively. Therefore Jeo is given by Jeo = v Ci
ð8Þ
where Ci is the average concentration of the ionic species i near the membrane. In the present study, the lidocaine transport efficiency increased with increasing voltage and duty cycle. This result suggests that the width of the square wave, as the DC component, plays an important role in the generation of electrorepulsion and the periodic polarity alteration with the introduction of the square wave is involved in electroosmosis. Electroosmosis is a phenomenon which an electrically driven flow of ions across a membrane with a net charge can induce the coupled flow of solvent [13]. A bulk motion of the solvent itself produced by electroosmosis carries ions or neutral species across a membrane, within the solvent stream. According to our previous studies [8–10], electrorepulsion and electroosmosis are closely involved in the transport of lidocaine with the application of AC. Yan and Li et al. accomplished successful transport of a charged permeant, tetraethyl ammonium, and a neutral permeant, arabinose, across a synthetic membrane by application of a square-wave AC iontophoresis. Their report suggested that both electrorepulsion and electroosmosis play essential roles in the transport of substances in AC fields, although the electroosmotic enhancement was smaller than the electrorepulsive effect [4]. Electroosmosis frequently occurs in DC fields, but can also be observed in AC fields. In the present study, not only electrorepulsion but also electroosmosis were probably involved in the enhancement of lidocaine transport caused by AC delivery with duty cycle. However, the relative contributions of electrorepulsion and electroosmosis to the total iontophoretic flux have been proven difficult to quantify, due to the difficulty of designing appropriate experiment. The relative importance of electrorepulsion and electroosmosis depends on the physiochemical and electrical characteristics of the membrane and of the permeant. 4.3. pH changes and the electrode It has been reported that various physical and chemical properties of the donor substances, such as the concentration of the substance, pH, dissociation state of ions, molecular size, molecular weight, charge state, lipid solubility, water solubility and chemical stability, influence the
321
transport efficiency [13]. Previous reports have described that both the pH environment of the skin or solution and the degree of drug ionization are involved in iontophoretic drug transport [16,17]. In the present study, the pH of the solution in the receptor chamber showed duty cycle-dependence under the application of 10, 20 and 30 V. Voltagedependence of the pH was also observed at duty cycles of 60%, 70%, 80%, 90% and 95%. These findings indicate that the water in the chamber was electrolyzed, resulting in the production of OH− ions in the receptor chamber. The reaction rate of electrolysis increased depending on the voltage and the duty cycle. The production of ions may reduce the flux of similarly charged solute ions. On the other hand, H+ ions produced by electrolysis would compete with the positively charged lidocaine ions under such circumstances, resulting in decrease of the iontophoretic drug flux, because they carry a fraction of the total current. However, the pH decreased with time with the application of a duty cycle of 50% at each voltage and during passive diffusion. The pH decrease associated with passive diffusion indicates that the dissociated lidocaine hydrogen ion (C14H22N2O∙H+) spontaneously permeated from the donor chamber to the receptor chamber without water electrolysis, resulting in the accumulation of H+ ions. The pH decrease observed with a 50% duty cycle was due to accumulation of dissociated lidocaine hydrogen ions transported by the application of an electric field, and suggests a lower degree of water electrolysis. The electrode materials affect the pH of the solution. In the present study, platinum electrodes were used in order to prevent the generation of competing ions. The electrodes used for iontophoresis are classified roughly into two categories, namely, the inert type and the reversible type. The inert-type electrodes, such as platinum and aluminum, do not take part in electrochemical reactions, but cause electrolysis of water, resulting in the production of H+ at the anode and OH− at the cathode. On the other hand, the reversible- type electrodes, such as silver/silver chloride (Ag|AgCl) electrodes, are resistant to pH changes, because of the oxidation reaction of the electrodes expressed as follows. Ag þ Cl− ¼ AgCl þ e−
ð9Þ
This oxidation reaction of the electrode produces insoluble silver chloride, which precipitates on the anode surface, resulting in the absence of net migration of silver ions into the donor chamber. However, the reversible-type electrodes generate competing ions resulting in a decrease of the transport efficiency [13]. 5. Conclusion Our experimental system was successfully used to evaluate the effect of the waveform component of high-frequency AC voltage on lidocaine transport across a cellophane membrane. The results of this study indicated that lidocaine was transported efficiently through a cellophane membrane with the use of square-wave AC iontophoresis with a duty cycle, in a voltage-, time- and duty cycle-dependent manner. With the application of 30−V, 1 kHz AC voltage with a 95% duty cycle for 60 min, the lidocaine concentrations were about twofold higher than that observed with the application of a 50% duty cycle and about threefold higher than that observed following passive diffusion, however, the pH of the solution in the receptor chamber increased markedly because of water electrolysis. When an AC voltage of 10 V was applied with a 70% duty cycle or of 20 V was applied with a 60% duty cycle, lidocaine was transported more efficiently as compared with that observed with a 50% duty cycle, with also a smaller change of the pH of the solution in the receptor chamber. Consequently, it is suggested that application of 10 V with a 70% duty cycle or 20 V with a 60% duty cycle is optimal for lidocaine transport in clinical situations, because of the lesser degree of water electrolysis as compared with that observed with the application of 30 V with a 95% duty cycle.
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