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Study of diffusion characteristics of salicylic acid through cellulose acetate membrane and extracted mouse skin by iontophoresis.

The diffusion of salicylic acid, a model drug, loaded in prepared corn starch paste was studied through cellulose acetate membrane and extracted mouse skin by the application of iontophoresis. Current was applied from a custom designed current delivery circuit. The in-vitro experiments were conducted using modified Franz diffusion cell with stainless steel (Grade 316L) electrodes. Pulsed DC square-wave with different pulse durations was used as current profile with a maximum current density of 0.4mA/[cm.sup.2]. As expected, it was observed that the rate of drug diffusion varied almost nearly with the applied current density. The amount of drug diffusion varied with the current profile in case of cellulose acetate membrane. The maximum flux of salicylic acid obtained was 0.0015 mg/[cm.sup.2]/s through cellulose acetate membrane and 4.9e-5 mg/[cm.sup.2]/s in case of extracted mouse skin. [C] Society for Biomaterials and Artificial Organs (India), 20070704-1228.

Introduction

Transdermal delivery of drugs offers a number of advantages over other methods of drug delivery. The advantages include, noninvasive delivery, it avoids the first pass effect experienced by drugs delivered orally and chemical degradation of the drug in the potentially hostile environment of the gastrointestinal tract. Transdermal delivery has its own disadvantages which limit the range of drugs that can be delivered transdermally. The human skin consists of three main layers namely epidermis, dermis and hypodermis. The thickness of the epidermis layer varies from 0.06 mm on eyelids to 0.8 mm on soles of feet and palms. The major barrier to permeation of drugs across the skin is the stratum corneum (approximately 10-20 [micro]m thick) which prevents water loss from the skin and protects the body from the outside environment. The stratum corneum layer of skin is composed of multi-lamellar lipidic structure punctuated by dead proteinaceous corneocytes that impose a significant tortuosity on the diffusion path across the membrane [1]. Various skin penetration enhancing techniques has been developed such as chemical enhancers, iontophoresis, sonophoresis (phonophoresis) and electroporation to overcome this formidable barrier.

Iontophoresis is the application of an electric current which enhances the delivery of ionized as well as unionized drug molecules through any synthetic or biological membrane like skin [2-4]. By application of iontophoresis the range of drugs delivered transdermally can be extended. The advantages of iontophoresis include, enabling continuous or pulsatile delivery of drugs, control over the amount of drug delivered by regulating the current passing through the system [5], easier termination of drug application and ability for systemic or local delivery [6]. However, there are a few disadvantages like skin irritation [6] and a handful number of drugs (mainly low molecular weight) applicable by iontophoresis.

Since several drugs belonging to the non-steroidal anti-inflammatory drugs (NSAID) category provoke harmful effects in the gastrointestinal tract after oral administration, transdermal iontophoretic delivery of these drugs needs to be investigated [7]. Our present work is the study of in-vitro iontophoretic diffusion of salicylic acid (SA), an NSAID, through cellulose acetate membrane and extracted mouse skin by a custom designed low-cost portable current delivery device. The effect of applied current density and various current profiles through the membranes were studied. Starch, obtained from natural resources, is widely used in food and non-food industries like pharmaceutical, paper and textile industries [8, 9]. 'Cooking' starch together with water results in gelatinization. Crystalline structures of starch molecules are disrupted by increased hydrogen bonding between water molecules and hydroxyl groups, and this induces granular swelling. At the same time, amylase molecules diffuse out of the swollen starch granules [10]. Gelatinized starch is, in essence, an amylose gel network with the swollen granules as filler [11]. In the present study, starch paste was used as drug carrier for the iontophoretic system. The drug (SA) was loaded in prepared corn starch paste and was placed between the active electrode and the diffusion membrane. The experiments were performed using modified Franz diffusion cell with stainless steel (grade 316L) electrodes.

The stratum corneum of the skin shows two important electrical features: first, it tends to become polarized as an electric field is continuously applied, second, its impedance changes with the frequency of the applied electric field [12-14]. When an electric field with DC (direct current) is applied in a continuous manner to the skin, an electrochemical polarization develops rapidly and it often operates against the applied electric field and readily decreases the magnitude of effective current across the skin, and so the current gradient through the skin decays exponentially. Consequently, the efficiency of iontophoresis-facilitated delivery is reduced as a function of the duration of dc iontophoresis treatment. This polarization can be overcome by using pulsed DC, a direct current delivered periodically. During the "off time" of the waveform the skin becomes depolarized and returns to its initial unpolarized status. Moreover, pulsed DC reduces the total current quantity passing through the skin diminishing the risk of skin alterations like burns and irritation [15]. For the experimentations pulsed DC square-wave with different pulse durations and 50% duty cycle was used. For most of the experiments, the maximum current density was kept at 0.4 mA/[cm.sup.2], which is below the recommended safe level of 0.5 mA/[cm.sup.2] for transdermal drug delivery [16].

Materials and Methods

Chemicals

Salicylic acid (SA) was obtained from Loba-Chemie Co., Mumbai, India. Buffer capsules were obtained from Merck Limited, Mumbai, India. Cellulose acetate paper (Grade 40) was obtained from Whatman, India. Corn starch (CS) was obtained from Sigma, India. Double distilled water was used throughout the study.

Preparation of corn starch paste

Corn starch paste was used as a medium for the drug (SA) application over the membranes for iontophoresis. For this purpose 90 ml of distilled water was heated to boiling and to it 1g salicylic acid was added by stirring. Separately, 10g of corn starch was added in 10 ml of distilled water and this suspended solution was added to the boiling aqueous solution of salicylic acid. The mixture was 'cooked' for 5 minutes after swelling. The paste thus prepared was left for cooling before being used for the experiments.

Skin preparation

Hair was shaved by a razor from the excised mouse skin. The adherent fat and other visceral debris from the under surface were removed by scalpel and isopropyl alcohol. Then it was washed in tap water. Finally the skin was soaked in 0.9% saline water solution and preserved at 4[degrees]C before further experimentation.

Circuit components

IC LM324 was obtained from SGS Thomson Microelectronics, IC HEF4011B was obtained from Philips Semiconductors, IC 555 was obtained from SGS Thomson Microelectronics.

Waveforms used

For various experiments, pulsed dc square-wave of different pulse durations (0.25s, 1s, 2s and 4s) and with 50% duty cycle were used. The different waveforms were taken from the various points in the custom designed current delivery circuit as shown in Fig. 1.

Circuit design

The circuit is based on a low power quad Operational amplifier IC (IC LM324), with a 555 timer and a quadruple 2 input NAND gate (IC 4011). The schematic representation of the circuit is shown in Fig. 1. Opamp Q1 is used as a multivibrator for generating [+ or -] 10V square wave output. The positive half (a) of the waveform is AND-ed with a higher frequency (adjustable) 10V square wave pulse train obtained from the 555 Timer (d). Now this waveform (d) is added to the negative part of the squarewave (b) generated from Q1 by Opamp Q2 which is used as an inverting summing amplifier to obtain waveform (c). The pulse width was adjusted by the potentiometer at Q1. Different waveforms (a, b, c, d) used in the experiments were taken from various points in the circuit through manual switches. The amplitude of the current delivered was adjusted by a potentiometer connected in series with the load. The waveforms were monitored by a monitor measuring the voltage drop across a 1 Ohm resistor placed in series with the load. [+ or -] 12V power supply was obtained by a 230-12V step-down transformer connected to 230V, 50Hz power supply line.

[FIGURE 1 OMITTED]

Diffusion experiments

In-vitro diffusion experiments were carried out using modified Franz's diffusion cell which was custom designed. It had a receptor compartment volume of 54 ml and the surface area available for diffusion was 2.7 [cm.sup.2]. A narrow sampling terminal was connected to the receptor compartment. The schematic representation of the experimental setup is shown in Fig. 2. The stainless steel electrodes were cut into the required size and shape from stainless steel sheets. The active electrode was 0.11 mm thick and had a surface area of 2.7 [cm.sup.2].

The receptor compartment was filled with 0.9% saline water solution and the pH was kept at 7.4 [+ or -] 0.05. During the experiments the receptor fluid was continuously stirred by a magnetic stirrer. The experiments were done at room temperature (27[degrees]C). The donor compartment consisted of drug (SA) loaded corn starch paste. The active electrode was placed above the drug loaded corn starch paste and the reference electrode was placed at the reference terminal.

The diffusion studies were carried out through cellulose acetate paper and excised mouse skin. At predetermined times (5, 10, 15, 20, 30 and 45 minutes), 1 ml samples were withdrawn from the receptor compartment through the sampling terminal and were immediately replaced by the same volume of drug free buffered saline solution. These samples were used to determine SA concentration for the subsequent calculation of the diffusion parameters. The samples were analyzed by UV Spectrophotometer. The reduction in the concentration of the remaining solution in the donor chamber was taken into account for the subsequent calculations.

The apparent instantaneous flux of the permeant (here SA) between each time points were determined by [17]:

F =/(1 dQ)/A dt (1)

Where Q is the cumulative amount transported across the membrane, t is the time, A the diffusion surface area.

[FIGURE 2 OMITTED]

Results and discussion

Effect of current density

[FIGURE 3 OMITTED]

Fig. 3 shows the percentage release of SA through cellulose acetate membrane as a function of applied current amplitude (and hence current density, as the diffusion area is unchanged). The data shows the percentage of SA present in the SA loaded corn starch paste that diffused through the membrane in 45 minutes at various current densities. Here the effect of passive diffusion was subtracted from the iontophoretic diffusion after calculating the passive diffusion separately under identical conditions. As expected, the percentage diffusion varies almost linear with the increase in the applied current density [7]. Each experiment was conducted for 45 minutes. The data point's represents mean of three determinations.

Effect of various waveforms

Passive diffusion of SA through cellulose acetate membrane was compared with iontophoretic diffusion by various current profiles. The effect of varying the current profile was studied by applying pulsed dc with different pulse widths. The apparent flux for pulse train of different pulse widths is shown in Table 1. The apparent flux was calculated from Eq. 1.

From the data obtained it is apparent that the dc square-wave of pulse width 1sec shows comparatively better results than the other waveforms in terms of average flux (Table 1) and linearity with respect to time. This could be attributed to the fact that with the increase in the pulse duration the effect of polarization becomes more prominent. In all the cases the current density was kept at 0.4 mA/[cm.sup.2] and drug was loaded in corn starch paste. The experiments were conducted at room temperature.

[FIGURE 4 OMITTED]

Diffusion study through mouse skin

Drug (SA) loaded corn starch paste was prepared and applied over mouse skin. Flux of SA through mouse skin was observed to be much lower than that through the cellulose acetate membrane. This is due to the fact that the stratum corneum layer of the skin with its multilamellar lipidic structure poses significant barrier to the flow of SA through it. It is observed that initially there is a higher rate of drug release which gets reduced gradually with time. The reason behind this may be that initially there is a higher difference in concentration of SA between the donor and receiver chamber of the system which gradually gets reduced with more and more drug diffusing to the receiver chamber.

Unlike diffusion through cellulose acetate membrane, there was no significant difference in the rate of diffusion through the skin with variation in the current profile. The reason behind this could be that, once an iontophoretic diffusion path through the stratum corneum of the skin has been established, the current profile doesn't govern the flow rate of the drug.

The skin showed minor superficial changes in colour at the site of the electrode after 45 minutes of iontophoresis at a current density of 0.4mA/[cm.sup.2]. However no significant variation in the texture of the skin surface was found when observed under an optical microscope.

[FIGURE 5 OMITTED]

Conclusion

In the present study, a custom designed low-cost portable current delivery device was used to analyze the diffusion characteristics of SA through cellulose acetate membrane and extracted mouse skin. As expected there was a linear increase in percentage diffusion of SA with the increase in the applied current density. While the square-wave dc pulse train with 50% duty cycle showed a better flux of drug with respect to the other waveforms through cellulose acetate membrane, there was not much difference in the drug flux with variation in current profile through extracted mouse skin. Different other waveforms need to be studied to come to any conclusion on this. The iontophoretic current delivery device, being made of low cost & easily available components, can be easily assembled on a XcmXYcm PCB.

Received 29 August 2007; published online 18 December 2007

References

[1.] R.O. Potts and M.L. Francoeur, The influence of stratum corneum morphology on water permeability, J. Invest. Dermatol. 96 (1991) pp. 495-499.

[2.] G.B. Kasting, Theoretical models for iontophoretic delivery, Adv. Drug Deliv. Rev. 9 (1992) pp. 177-199.

[3.] J.B. Phipps and J.R. Gyory, Transdermal ion migration, Adv. Drug Delivery Rev. 9 (1992) pp. 157-176.

[4.] M.J. Pikal, The role of electroosmotic flow in transdermal iontophoresis, Adv. Drug. Deliv. Rev. 9 (1992) pp. 201237.

[5.] R.R. Burnette and D. Marrero, Comparison between the iontophoretic and passive transport of thyrotropin releasing hormone across excised nude mouse skin, J. Pharm. Sci. 75 (1986) pp. 738-43.

[6.] Y. Wang, R. Thakur, Q. Fan and B. Michniak, Transdermal iontophoresis: combination strategies to improve transdermal iontophoretic drug delivery, Euro. J. of Pharm. and Biopharm. 60 (2005) pp. 179-191.

[7.] Y.N. Kalia, A Naik, J Garrison and R.H. Guy, Iontophoretic drug delivery, Adv. Drug Delivery Rev. 56 (2004) pp. 619-658.

[8.] P.N. Bhandari and R.S. Singhal, Effect of succinylation on the corn and amaranth starch pastes, Carbohydrate Polymers 48 (2002) pp. 233-240.

[9.] J.Y. Thebaudin, A.C. Lefebvre and J.L Doublier, Rheology of Starch Pastes from Starches of Different Origins: Applications to Starch- based Sauces, Lebensm.-Wiss. u.-Technol. 31 (1998) pp. 354-360.

[10.] R.L. Whisler and J.N. Bemiller, Carohydrate chemistry for food scientists, St. Paul, Minnesota: American Association of Cereal Chemists (1997) pp. 117-151.

[11.] M.J. Miles, V.J. Morris, P.D. Orford and S.G. Ring, The role of amylose and amylopectin in the gelation and retrogradation of starch, Carbohydrate Research 135 (1985) pp. 271-281.

[12.] T. Yamamoto and Y. Yamamoto, Analysis for the change of skin impedance, Med. Biol. Eng. Comput., 15 (1977) pp. 219.

[13.] K. Okabe, H. Yamaguchi and Y. Kawai, New iontophoretic transdermal administration of the beta blocker metoprolol, J. Controlled Release, 4 (1986) pp. 79.

[14.] Y.W. Chien, P. Lelawongs, O. Siddiqui, Y. Sun, W.M. Shi, Facilitated transdermal delivery of therapeutic peptides and proteins by iontophoretic delivery devices, J. Controlled Release, 13 (1990) pp. 263-278.

[15.] S. Thysman, V. Preat and M. Roland, Factors affecting iontophoretic mobility of metoprolol, J. Pharm. Sci. 81 (1992) pp. 670-675.

[16.] P. Singh and H.I. Maibach, Iontophoresis in drug delivery: basic principles and applications, Crit. Rev. Ther. Drug Carrier Syst. 11 (1994) pp. 161-213.

[17.] G. Yan, S.K. Li and W.I. Higuchi, Evaluation of constant current alternating current iontophoresis for transdermal drug delivery, J of Control. Release 110 (2005) pp. 141-150

[18.] Mosmann T., Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays, J Immunol Methods 65 (1983), pp. 55-63.

Rajdeep Dasgupta (#), Ajit Kumar Banthia (#) (@), D.N. Tibarewala *

(#) Materials Science Centre, Indian Institute of Technology, Kharagpur 721302, India

* School of Bioscience and Engineering, Jadavpur University, Kolkata, India

(@) corresponding author e-mail: ajitbanthia2000@yahoo.co.in
Table 1: Apparent flux for pulse train of different
pulse widths

 Drug flux
Waveform (mg/[cm.sup.2]/s)

1s pulse width 0.0015
2s pulse width 0.0009
4s pulse width 0.007
0.25 with 1s interval
 of no current 0.0006
" Passive 0.0002

Table 2:

Waveform Drug flux
 (mg/[cm.sup.2]/s)

1s pulse width 4.90e-5
2s pulse width 4.21e-5
4s pulse width 4.00e-5
0.25 with 1s interval
 of no current 3.39e-5
" Passive 1.20e-5
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Author:Dasgupta, Rajdeep; Banthia, Ajit Kumar; Tibarewala, D.N.
Publication:Trends in Biomaterials and Artificial Organs
Geographic Code:9INDI
Date:Jan 1, 2008
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