J Med Dent Sci 2014； 61： 41－48

Iontophoresis (IOP) is a noninvasive method of delivering medication transcutaneously through the skin. The electrodes used in this method should tightly fit to rough and irregular surfaces and be biologically safe, easy to handle and prepare, and cost-effective. To satisfy these requirements, calcium alginate gel can be a candidate electrode for IOP. Using calcium alginate gel electrodes, we examined whether lidocaine can be effectively transported across an excised rat skin by square-wave alternating current (AC) application. A square-wave AC with either a 70 % or 80 % duty cycle was continuously applied to 0.5 % calcium alginate gel electrodes containing 10 % lidocaine at 10 V and 1 kHz for 60 min. Lidocaine concentration was measured using a spectrophotometer and the temperature of the gel was determined. The lidocaine concentrations for AC-IOP at the 70 % and 80 % duty cycles were significantly higher than that without AC-IOP. Furthermore, the group with the 80 % duty cycle showed higher lidocaine concentrations than the group with the 70 % duty cycle. The temperatures of all the groups were lower than 28 ℃ throughout the procedure. In conclusion, the calcium alginate gel can be used as a possible matrix for IOP electrodes.

Key words: Drug delivery system (DDS), Iontophoresis (IOP), Calcium alginate gel, Alternating current (AC), Electrode

1. Introduction

  Iontophoresis (IOP) is a noninvasive method using a drug delivery system (DDS), by which ionic compounds or noncharged drug molecules are delivered using an external electric field. IOP enhances the transport of ionic substances across membranes or the skin via principal mechanisms, electrorepulsion and electroosmosis1,2. Electrorepulsion is considered to play the most crucial role in the transport of ionic drugs through a membrane or the skin. Transdermal drug delivery using IOP has a number of potential advantages; drug degradation through the stomach and intestine and the first pass through the liver are completely avoided, and in particular, it is painless. IOP has two types, IOP using direct current (DC) and that using alternating current (AC). Generally, DC has been widely used because its transport efficiency is higher than that of AC. The DC-IOP of anesthetics such as lidocaine is used in clinical settings. However, it has some side effects such as electrical or chemical burns, and erythema due to electrode polarization during electrolysis. The authors of the present paper and their colleagues have modified a DDS using AC-IOP for safer and more effective application associated with fewer side effects3-12. Our previous study, Kinoshita et al. found that 10 % lidocaine could be transported through excised rat skin in vitro using a sinusoidal AC voltage4. Although their report used sinusoidal AC, excised rat skin was also applied as our study. Hayashi et al.

Corresponding Author: Tomoko EbisawaSection of Anesthesiology and Clinical Physiology, Department of Oral Restitution, Division of Oral Health Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, JapanE-mail: tomanph@tmd.ac.jpReceived December 9, 2013；Accepted March 14, 2014

Original Article

Evaluation of calcium alginate gel as electrode material for alternating current iontophoresis of lidocaine using excised rat skin

Tomoko Ebisawa1), Atsushi Nakajima1), Haruka Haida1), Ryo Wakita1), Shizuka Ando1), Tomohiko Yoshioka2), Toshiyuki Ikoma2), Junzo Tanaka2) and Haruhisa Fukayama1)

1) Section of Anesthesiology and Clinical Physiology, Department of Oral Restitution, Division of Oral Health Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University2) Department of Metallurgy and Ceramics Science, Graduate School of Engineering, Tokyo Institute of Technology

42 J Med Dent SciT. Ebisawa et al.

indicated that lidocaine was transported efficiently through a cellophane membrane using square-wave AC-IOP with a duty cycle, in a voltage-, time- and duty cycle-dependent manner8. Furthermore, the 60 % at 20 V or 70 % at 10 V duty cycle are good conditions for transport8. They applied a bipolar square wave with a duty cycle which is combined with DC8. Wakita et al. reported that the clinical application of lidocaine by IOP using 70 % and 80 % duty cycles provided a significant anesthetic effect in comparison with the 60 % duty cycle10. Haida et al. set the duty cycle at 80 % and at 2.5 V and 5 V for transport energy11. Therefore we applied 70 % and 80 % duty cycles at 10 V. When applying IOP, matrices of electrodes are also very important for effective drug delivery. As electrodes for IOP, they should tightly fit even to rough and irregular surfaces and be biologically safe, easy to handle and prepare, and cost-effective. To satisfy these requirements, calcium alginate gel can be a candidate electrode for IOP11,13,14. Haida et al. found that lidocaine can be released from calcium alginate gel used as a matrix for IOP electrodes by square-wave AC application through a cellophane membrane11. We proceeded to apply their method to clinical settings. The goal of our study was to determine whether calcium alginate gel can be used as electrodes in AC-IOP with a duty cycle through an excised rat skin by examining lidocaine transport.

2. Materials and Methods

  The experiment protocol was approved by the Institutional Animal Care and Use Committee (Center for experimental Animal) of Tokyo Medical and Dental University (No. 0130055A). All experimental procedures were performed in accordance with the guidelines of the above-mentioned review board.

2.1. Materials  2.1.1 Animals  Eight-week-old male Wistar/ST rats weighing 365 - 490 g were purchased from Sankyo Laboratory Service (SLC, Tokyo, Japan). All animals were housed in an air-conditioned room until experiment. Thirty rats were sacrificed for the experiment. Abdominal hair was shaved the day before the experiment. The rats were anesthetized by peritoneal injection of sodium pentobarbital (100 mg/kg body weight) (Somunopentyl, Kyoritsu Seiyaku Corporation, Tokyo, Japan) and then sacrificed12. Overdose administration of sodium pentobarbital (100 - 120 mg/kg) is applicable to many

species of animals such as mice, rats, rabbits, dogs, pigs and monkeys. The skin was excised and the subcutaneous fat tissues were removed. The excised abdominal skin was composed of the stratum corneum, viable epidermis and upper serimis. The excised abdominal skin tissue was approximately 25 mm in diameter and 0.8 mm in thickness4.  2.1.2 Calcium alginate gel  Sodium alginate (300 - 400 cP, Wako Pure Chemicals Industries Co., Ltd., Osaka, Japan) was used in the experiment. Distilled water (resistivity > 18 MΩ・cm) was prepared using Milli-Q SP TOC (Merck Millipore, Billercia, MA, USA).   An alginate aqueous solution of 1 % was prepared using distilled water to obtain 0.5 % calcium alginate gel. Fifty milliliters of 0.01 M CaCl2 (FW, 110.99; Sigma-Aldrich Co., Ltd., St. Louis, USA) aqueous solution was gradually poured into a 50 ml alginate solution with stirring. The resulting viscous alginate solution was stored at 4 ℃ for 12 h. Then, another 100 ml of 0.01 M CaCl2 aqueous solution was slowly added to the viscous solution. The CaCl2 and viscous alginate solutions were completely separated. The 0.01 M CaCl2 aqueous solution was exchanged for 0.1 M CaCl2 aqueous solution until it completely gelled at 4 ℃. The obtained calcium alginate gel was cut into a discus shape (20 mm in diameter, 2 mm in thickness). The discus-shaped gel was washed and stored in distilled water until the experiment. In the experiment, it was prepared depending on the alginic acid concentration of the gel, namely, 0.5 % (Figure 1). Haida et al.11 showed that 0.5 % alginic acid gel was optimal for lidocaine transport by AC-IOP, indicating that lidocaine was efficiently transported through a cellophane membrane using a square wave. Therefore our study used 0.5 % alginic acid gel.

  2.1.3 Lidocaine hydrochloride solution  Lidocaine hydrochloride (C14H22N2O∙HCl: FW, 270.8; H2O content, 1 mol/mol) was purchased from Sigma-

Fig. 1. 0.5 % calcium alginate gel.

43Calcium alginate gel as electrode for AC IOP

Aldrich Co., Ltd. (St. Louis, Mo, USA). Ten percent lidocaine hydrochloride was prepared using distilled water for experiments. The pH of 10 % lidocaine hydrochloride solution was 5.44.  2.1.4 Structure and setup of experimental cell  A cylindrical acryl drug delivery cell consisting of two chambers (one was the donor chamber and the other was the receptor chamber) was constructed. Platinum plate electrodes of 20 mm diameter and 0.5 mm thickness were attached to the opposite ends of the two chambers of the cell. The freshly excised rat abdominal skin was sandwiched between the two chambers. The distance from a platinum plate electrode of the receptor side was 10 mm, and the distance from a platinum plate electrode of the donor side was 2 mm. The donor chamber was filled with calcium alginate gel soaked in 10 % lidocaine hydrochloride solution for 1 h, whereas the receptor chamber with a capacity of 3.1 cm3 was fi l led with 3.0 ml of disti l led water or phosphate-buffered saline (contents: sodium phosphate dibasic, 0.162 M; sodium phosphate monobasic, 0.038 M; sodium chloride, 1.5 M, pHs, 7.3 - 7.5) (Figure 2). Acryl cells were set at room temperature (24.0 ℃).

2.2 Methods  Our methods and measurements basically followed

those previously reported (Kinoshita et al.4, Hayashi et al.8 and Haida et al.11) with slight modifications.  2.2.1 Electric current application  To examine the relationship between the efficiency of lidocaine transport and the duty cycle, a bipolar square wave with two different duty cycles, either 70 or 80 % (Figure 3), was continuously applied at a voltage of 10 V and a frequency of 1 kHz between the parallel platinum electrodes of the drug delivery cell for 60 min using a function/arbitrary waveform generator (Agilent 33250A, Agilent Technologies, Colorado, USA) and a

Fig. 2. Setup of experimental system. Consisting of a drug delivery cell with the donor and receptor chambers, a thermocouple microprobe and a thermostat, with a function generator and a high-speed power amplifier.

Fig. 3. Diagram of bipolar square wave (AC) with 70 % or 80 % duty cycle. The duty cycle is the ratio of the positive cycle to the full cycle.

44 J Med Dent SciT. Ebisawa et al.

high-speed power amplif ier (4025, NF Electric Instruments, Kanagawa, Japan). For the passive diffusion group, no current was applied. The waveform and output voltage were monitored using a digitizing oscilloscope (HP54503A, Hewlett Packard, Tokyo, Japan) throughout the experiment.

2.3 Measurement  2.3.1 Lidocaine concentration  Measurement system was referred to Hayashiʼs protocol8. Twenty microliters of lidocaine hydrochloride solution was sampled every 10 min during a 60 min AC application using a micropipette (20 - 200 μl, Nichiryo, Tokyo, Japan) placed at the center of the receptor chamber. The samples were diluted 25-fold with distilled water or phosphate-buffered saline. 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, Ltd., Tokyo, Japan) at room temperature. The absorbance of the samples was measured at a wavelength of 262 nm (Ultraviolet light) and an optical path of 10 mm. The specific wave length of lidocaine's absorbance is 262 nm. Lidocaine concentration was quantified using a calibration curve. The detection limit was 11.6 μg/ml4.  2.3.2 Temperature  A thermocouple microprobe (BAT-12, Physitemp, NJ, USA) was inserted directly into the center of the donor chamber to measure the temperature of the gel. Temperature was monitored every 10 min during a 60 min AC application.

  2.3.3 pH  Hayashi et al. measured pH, which showed below 9.6 under the same condition as ours. Therefore pH measurement was not performed in our study.

2.4 Statistical analyses  All values are presented as mean ± S.D. (n = 5). Two-way analysis of variance (ANOVA) was carried out to analyze the time and duty cycle dependences of the transport efficiency of lidocaine. In addition, the Tukey test was used to analyze the time and duty cycle dependences of the transport efficiency of lidocaine, temperature changes, duty cycle, and solution of the receptor chamber. Statistical significance was assumed at p < 0.05. All statistical analyses were performed using Kyplot 5.0 (KyensLab Inc., Tokyo, Japan).

3. Results

3.1 Lidocaine concentration changes  3.1.1 Changes in lidocaine concentration in

receptor chamber containing distilled water  Figure 4(a) shows the changes in l idocaine concentration in samples in the receptor chamber containing distilled water induced by the application of a square-wave AC with three different duty cycles, namely, 70 % (the 70 % group) and 80 % (the 80 % group) at 10 V and 1 kHz, and with no current application (the passive diffusion group) for 0.5 % alginic acid gel. In all the groups, the concentration of lidocaine transported from the calcium alginate gel to

Fig. 4. Changes in lidocaine concentration in 0.5 % alginic acid gel groups with AC square-wave application with 2 different duty cycles and without duty cycle at 10 V and 1 KHz. (a) Distilled water in chamber and (b) phosphate-buffered saline in chamber. The error bars represent the standard deviations from the mean. ＊ indicates a statistically significant difference among the conditions.

(a) (b)

45Calcium alginate gel as electrode for AC IOP

the receptor chamber significantly increased in a time-dependent manner. In the 70 % group, the lidocaine concentration was significantly higher 40, 50 and 60 min after IOP than in the passive diffusion group. Furthermore, in the 80 % group, the concentration was significantly higher during 10 - 60 min after the start of IOP than in the passive diffusion group. The 80 % group showed significantly higher concentrations at 10, 40 and 60 min than the 70 % group. At 60 min application of IOP, the concentration was 3.69 ± 1.74 (mean ± SD) mmol/l, which was twice that in the 70 % group (1.72 ± 0.37 mmol/l), and about 4.7 times higher than that in the passive diffusion group (0.79 ± 0.41 mmol/l).  3.1.2 Changes in lidocaine concentration in

receptor chamber containing phosphate-buffered saline

  Figure 4(b) shows the changes in l idocaine concentration in the receptor chamber containing phosphate-buffered saline, instead of distilled water, in the 70 % group, 80 % group and passive diffusion group. The lidocaine concentration significantly increased with time in all the groups.  Compared with the passive diffusion group, the 70 % group showed higher concentrations of lidocaine during 30 - 60 min after the start of application; similar results were obtained in the 80 % group during 10 - 60 min. Significant differences were observed during 20 - 60 min between the 70 % group and the 80 % group.  At 60 min after the start of current application, the mean lidocaine concentrations were 1.55 ± 0.24 mmol/l in the passive group, 3.48 ± 0.66 mmol/l in the 70 %

group and 8.85 ± 1.26 mmol/l in the 80 % group. Therefore, the concentration in the 80 % group was about 2.5 times higher than that in the 70 % group and about 5.7 times higher than that in the passive diffusion group.

3.2 Temperature changes of alginic acid gel  3.2.1 Temperature changes of gel in receptor

chamber containing distilled water  Figure 5(a) shows that the temperature of 0.5 % alginic acid gel changed under the three IOP conditions. The gel temperature of the passive diffusion group was 22.68 ± 0.89 ℃ on average at the beginning, then it slightly increased to 22.86 ± 0.75 ℃ 60 min later. The temperature did not significantly change in the passive diffusion group (10 - 60 min). In the 70 % group, the gel temperatures ranged from 22.26 ± 1.4 ℃ (0 min) to 24.18 ± 0.66 ℃, which was significantly higher. The temperature started at an average of 23.62 ± 1.55 ℃ in the 80 % group, and slightly increased to 24.52 ± 1.36 ℃ 60 min later, which was not a significant difference. As compared with the gel temperature of the passive diffusion group (0 min), the temperature significantly increased at 40 min in the 70 % group and at 20 - 60 min in the 80 % group. There were no significant differences in temperature between the 70 % group and the 80 % group. The temperatures of the gel ranged from 22 to 25 ℃ throughout the procedure.  3.2.2 Temperature changes of gel in receptor

chamber containing phosphate-buffered saline  Figures 5(b) shows the changes in the temperature

(a) (b)

Fig. 5. Changes in temperature of 0.5 % alginic acid gel. (a) Distilled water in chamber and (b) phosphate-buffered saline in chamber. The error bars represent the standard deviations from the mean. ＊ indicates a statistically significant difference among the conditions.

46 J Med Dent SciT. Ebisawa et al.

of 0.5 % alginic acid gel in the three groups. The temperatures of the gel samples were from 21.84 ± 0.91 to 22.2 ± 0.35 ℃ in the passive diffusion group in the receptor chamber containing phosphate-buffered saline. The temperature did not change significantly during the procedure. In the 70 % group, the temperatures ranged from 22.22 ± 0.79 (0 min) to 24.34 ± 1.61 ℃ (60 min), and the temperature increase was significant at 40 and 60 min compared with the starting temperature. The temperature in the 80 % group changed from 23.9 ± 0.52 at the beginning to 26.8 ± 0.26 ℃ at 60 min, and a significant difference was observed during 10 - 60 min. When compared with the passive diffusion group at the beginning, the 70 % group (30 - 60 min) and 80 % group (20 - 60 min) showed significantly greater temperature changes. The temperature obtained from the 80 % group was significantly higher than that obtained from the 70 % group at 10 and 20 min.  In summary, the temperature in every group increased in a time-dependent manner; however, it changed only from 22 to 27 ℃, which was lower than 28 ℃ throughout the study.

4. Discussion

4.1 Calcium alginate gel as electrode for AC-IOP  The electrodes for IOP need to hold drugs, adhere to the skin or mucosa and be harmless and easy to handle. To achieve these requirements, many types of material have been applied, such as unwoven fabrics, canvas, felt, paper towel, or cotton gauze, which were connected to metal or alloy electrodes15. These materials should be filled with the required amount of a drug before application and should be completely sealed so as not to leak. The preparation of these materials is usually very difficult. These materials also hardly adhere to rough skin or mucosa because they need flat surfaces. Skin irritation or rash is also a concern after long application. Other hydrophilic materials that can hold drugs have also been introduced as electrodes; they are poly(N-isopropylacrylamide), 2-hydroxyethyl methacrylate and polyvinyl alcohol16. They may cause inflammation of the skin and mucosa owing to their lack of biocompatibility and biodegradability. The use of the materials mentioned above may make it difficult to achieve our purposes. Therefore, we focused on calcium alginate gel, which is used for wound dressing, drugs, food, dental materials and cosmetic additives, because it is of natural origin and inexpensive. The gel can hold drugs and tightly fit even to rough and irregular wet

surfaces. These characteristics suggest that calcium alginate gel can be used as electrodes for IOP. Haida et al.11 previously showed that calcium alginate gel can be applied as a matrix for IOP electrodes. They reported that lidocaine was transported through a cellophane membrane using calcium alginate gel as IOP electrodes. Therefore, we examined in vitro whether lidocaine could be delivered across an excised rat skin, which is morphologically similar to the human skin, using calcium alginate gel electrodes. Our results indicate that lidocaine was successfully transported even across the excised rat skin.  Since we used calcium alginate gel soaked in 10 % lidocaine hydrochloride solution for 1 h before the procedure, its lidocaine concentration was not measured. A more concentrated solution may be preferable for a more rapid increase in lidocaine concentration.  Calcium alginate gel may shrink and become separated when a high voltage is applied, which causes insufficient IOP. Haida et al. suggested these possibilities at 70 % duty cycle and 10 V11. Our present study did not indicate any shrinkage and the gel can be applied as IOP electrodes.

4.2 Use of excised rat skin  Ideally, it is clinically most appropriate to use human skin or a three-dimensional cultured human skin to examine whether IOP can be applied in lidocaine transfer. However, neither of these two types of skin is always available. Moreover, the use of human tissues and organs has ethical problems and there is a quantitative restriction even if they are available. In addition, large variations of results were obtained in the case of using human skin specimens owing to differences in anatomical site, race, age, and gender17-19. Presently, an extracted laboratory animal skin is used as a substitute for human skin, and skin from the pig, mouse, guinea pig, and rat has been used17,20. It has been particularly reported that the skin of the pig has the sulcus, which is very similar to that in humans, less hair, and the horny cell layer similar in thickness to the human counterpart. However, the permeability coefficient tends to change depending on the area, and the skin is difficult to prepare accurately. Moreover, the data from in vivo reactions of drugs, toxicity and pharmacokinetics studies of the mouse and guinea pig are not sufficient for extrapolation to transdermal absorption in humans, compared with the rat. Therefore, the rat was selected in our study, and human transdermal absorption can be predicted in addition to easier handling of the specimen.

47Calcium alginate gel as electrode for AC IOP

  The skin has three layers, namely, the epidermis, dermis, and subcutaneous fat21,22. Generally, the skin-absorbed drug passes through the epidermis and dermis, is absorbed into the capillary vessels in the subcutaneous fat, and enters the systemic circulation22,23. Subcutaneous fat was excluded from the excised skin, and the epidermis and dermis were used in our study4,17-19.

4.3 Temperature changes of alginate gel electrode  According to previous reports, skin burn due to temperature increase is a concern in the cell, skin or mucosa following IOP application. Since Haida et al.1 did not measure the temperature of the cell, they set acryl cells in the thermostat, the temperature of which was maintained at 36.0 ℃. A temperature probe inside the calcium alginate gel was installed to determine the temperature in our setting. The temperature of the donor chamber was measured because the donor chamber is supposed to directly contact to the skin in clinical use. When using distilled water, its maximum temperature was 25 ℃, and less than 28 ℃ when using phosphate-buffered saline.  Generally, thermal burn in human skin occurs at more than 70 ℃ for 1 s or 44 ℃ for 6 to 10 h24,25. The results indicate that IOP using calcium alginate gel as the electrode can be safely applied to the skin without any burn.

4.4 Lidocaine concentration  Our study showed that the concentration of lidocaine transported from calcium alginate gels significantly increased in a time-dependent manner using an excised rat skin in all the groups. A comparison between the IOP groups and the passive group showed that the former groups had significantly higher lidocaine concentrations. In addition, the lidocaine concentration of the 80 % duty cycle group was significantly higher than that of the 70 % duty cycle group. The results indicate that lidocaine IOP using calcium alginate gel as electrodes was achieved under our specific conditions.  Our previous studies using a cellophane membrane revealed that lidocaine transport depends on duty cycle, which is similarly observed in our present study using an excised rat skin8,11. Haida et al. also showed that lidocaine concentration increased after 40 min of IOP and our present results revealed that it increased after 10 min of IOP11. The possible reasons for this increase are the voltages (5 and 10 V) and membranes (cellophane membrane and excised rat skin) used. Although it is difficult to compare the two

studies, it is considered that IOP in the present study was effectively performed regardless of the differences in the thicknesses of the cellophane membrane (0.036 mm) and excised rat skin (0.8 mm). Our comparison between the IOP groups and the passive group showed that the IOP groups had significantly higher lidocaine concentrations.  In percutaneous absorption, there are two routes, namely, the intra/trans-cellular route and appendage route. Although the horny barrier suppresses absorption through the intra/trans-cellular route, major transport is expected because of the large area of the skin. On the other hand, the appendage route directly utilizes pores and sweat pores for absorpt ion. Although the efficiencies of these routes are high, small amounts of substances can be absorbed because of the small area of the appendage route. These findings show that major percutaneous absorption depends on the intra/trans-cellular route15,22. IOP induces active transport owing to the small electrically resistant area in the appendage route, resulting in greater drug delivery in spite of the small area of the appendage route23,26. In addition to the intra/trans-cellular route, the appendage route activated by IOP enables greater transport than passive diffusion.

4.5 Duty cycles of 70 % and 80 %  Lidocaine transport induced by a bipolar square wave with 70 % and the 80 % duty cycles was significantly enhanced time-dependently compared with that by passive diffusion. Hayashi et al.8 showed that either 60 % duty cycle at 20 V or 70 % duty cycle at 10 V was optimal for lidocaine transport by IOP, indicating that lidocaine was efficiently transported through a cellophane membrane using a square wave. The results of their study indicate that lidocaine was transported efficiently in a voltage-, time- and duty cycle-dependent manner. However, a duty cycle of higher than 90 % increases pH8. Wakita et al. reported that the clinical application of lidocaine by IOP using 70 % and 80 % duty cycles provided a significant anesthetic effect10. Haida et al. set the duty cycle at 80 % and at 2.5 V and 5 V for transport energy11. The results of their study indicate that lidocaine was transported efficiently through a cellophane membrane by square-wave AC-IOP at a duty cycle, in a voltage- and time-dependent manner using calcium alginate gel11. Under our conditions, 70 % duty cycles was used to confirm that lidocaine was transported. After reconfirmation, 80 % duty cycles, which was more effective than 70 % duty cycles, was applied in our study. Therefore, we

48 J Med Dent SciT. Ebisawa et al.

set the optimal duty cycles to be 70 % and 80 % at 10 V. Although pH was not measured in our study, a duty cycle of higher than 90 % may increase pH.

4.6 Distilled water and phosphate-buffered saline  Under our conditions, distilled water was used to confirm that lidocaine was transported through a cellophane membrane. After reconfirmation, phosphate-buffered saline, which was more physiological than distilled water, was applied in our study.

5. Conclusions

  We confirmed that lidocaine was successfully released from calcium alginate gel used as a matrix for IOP electrodes by 80 % duty cycle square-wave AC appl icat ion through the excised rat skin. The temperature inside the gel was lower than 28 ℃ throughout the procedure, indicating that there is no risk of complications such as burns. These findings indicate that calcium alginate gel can be used as a matrix for IOP electrodes.

References1 Guy RH, Ka l ia YN, De lgado -Charro MB, e t a l .

Iontophoresis: electrorepulsion and electroosmosis. J. Controlled Release. 2000; 64: 129-32.

2 Rai R, Srinivas CR. Iontophoresis in dermatology. Indian J Dermatol. Venereol. Leprol. 2005; 71: 236-241.

3 Shibaji T, Yasuhara Y, Oda N, et al. A mechanism of the high frequency AC iontophoresis. J Control Release. 2001; 73(1): 37-47.

4 Kinoshita T, Shibaji T, Umino M. Transdermal delivery of lidocaine in vitro by alternating current. J Med Dent Sci. 2003; 50(1): 71-7.

5 Izumikawa H. Lidocaine transportation through a cellophane membrane by wide range AC frequencies. J Stom Sci. 2005; 72: 183-189. (in Japanese)

6 Haga H, Shibaji T, Umino M. Lidocaine transport through living rat skin using alternating current. Med Biol Eng Comput. 2005; 43(5): 622-9.

7 Ogami S, Hayashi S, Shibaji T, et al. Pentazocine transport by square-wave AC iontophoresis with an adjusted duty cycle. J Med Dent Sci. 2008; 55(1): 15-27.

8 Hayashi S, Ogami S, Shibaji T, et al. Lidocaine transport through a cellophane membrane by alternating current iontophoresis with a duty cycle. Bioelectrochemistry. 2009; 74(2): 315-22.

9 Wakita R, Oono Y, Ogami S, et al. The relation between epinephrine concentration and the anesthetic effect of lidocaine iontophoresis. Pain Pract. 2009; 9(2): 115-21.

10 Wakita R, Nakajima A, Haida Y, et al. The relation between the duty cycle and anesthetic effect in lidocaine

iontophoresis using alternating current. Pain Pract. 2011; 11(3): 261-6.

11 Haida H, Ando S, Ogami S, et al. In vitro evaluation of calcium alginate gels as matrix for iontophoresis electrodes. J Med Dent Sci. 2012; 59: 9-16.

12 Nakajima A, Wakita R, Haida H, et al. Efficacy of lidocaine iontophoresis using either alternating or direct current in hairless rats. J Med Dent Sci. 2013; 60: 63-71.

13 Saito N, Yoshioka T, Ikoma T, et al. Drug releasing properties of alginic acid layers electrochemically deposited on metal surfaces. In Proceedings of the 10th Asian BioCeramics Symposium 2010(ABC2010), Yogyakarta, Indonesia. Archives of BioCeramics Researches. 2010; 1(10): 110-113.

14 Kim YJ, Park HG, Yang YL, et al. Multifunctional drug delivery system using starch-alginate beads for controlled release. Biol Pharm Bull. 2005; 28: 394-397.

15 Costello CT, Jeske AH. Iontophoresis: Applications in transdermal medication delivery. Phys Ther. 1995; 75: 554-563.

16 Bhattarai N, Gunn J, Zhang MQ. Chitosan-based hydrogels for controlled, localized drug delivery. Adv Drug Deliv Rev. 2010; 62: 83-89.

17 Takeuchi H, Mano Y, Terasaka S, et al. Usefulness of rat skin as a substitute for human skin in the in vitro skin permeation study. Exp. Anim. 2011; 60(4): 373-384.

18 Hada N, Hasegawa T, Takahashi H, et al. Cultured skin loaded with tetracycline HCl and chloramphenicol as dermal delivery system: Mathematical evaluation of the cultured skin containing antibiotics. JCR. 2005; 28: 108: 341-350.

19 Ishii H, Todo H, Sugibayashi K. Effect of thermodynamic activity on skin permeation and skin concentration of triamcinolone acetonide. Chem. Pharm. Bull. 2010; 58(4): 556-561.

20 Barbero AM, Frasch HF. Pig and guinea pig skin as surrogates for human in vitro penetration studies: A quantitative review. Toxicol In Vitro. 2009; 23: 1-13.

21 Moschella SL, Hurley HJ. Dermatology volume 1, 2nd. USA, W.B. Saunders, 1985.

22 El Maghraby GM, Barry BW, Williams AC. Liposomes and skin: From drug delivery to model membranes. Eur. J. Pharm. Sci. 2008; 34: 203-222.

23 Sugino M, Todo H, Sugibayashi K. Skin permeation and transdermal delivery systems of drugs: history to overcome barrier function in the stratum corneum. Yakugaku Zasshi. 2009; 129: 1453-1458. (in Japanese)

24 Moritz AR, Henriques FC. Studies of thermal injury, II. The relative importance of time and surface temperature in the causation of cutaneous burns. Am J Pathol. 1947; 23(5): 695-720.

25 Despa F, Orgill DP, Neuwalder J, et al. The relative thermal stability of tissue macromolecules and cellular structure in burn injury. Burns. 2005; 31; 568-577.

26 Higo N. The recent trend of transdermal drug delivery system development. Yakugaku Zasshi. 2007; 127(4): 655-662. (in Japanese)