It is over several decades that ultrasound is used to enhance the transdermal drug delivery (phonphoresis). The mechanism of the enhancement is not fully understood and the ability of ultrasound on the enhancement for some drugs is unclear. The effect of continuous wave 870 KHz ultrasound at intensity of 1 W/cm2 for 15 minutes on transdermal absorption of piroxicam from solution and gel formulations in hairless rat skin was studied. Exposure to ultrasound increased the rate of diffusion from gel and solution of piroxicam to 10 and 3 times higher than that in skins not exposed to ultrasound. We strongly believe that the lower diffusion of piroxicam from the solution is caused by extra-bubbles generated by ultrasound. It can be suggested that cavitation activity and its negative surface charges play a dominant rule in phonophoresis. Copyright Â© 2006 by Razi Institute for Drug Research (RIDR)

بهلولی, شهاب

روانفر, رضوان

شیران, محمد باقر

متولیان, منیژه

English

ArUMS Digital Repository (سامانه اطلاعات زیست پزشکی و سلامت)

    
 IJPT  |  January 2008  |  vol. 7  |  no. 1  | 15-19 
RESEARCH ARTICLE 1 
The Effect of Bubble Surface Charge on 2 
Phonophoresis: Implication in Transdermal 3 
Piroxicam Delivery 4 
MOHAMMAD BAGHER SHIRAN, MANIJEH MOTEVALIAN, REZVAN RAVANFAR and SHAHAB 5 
BOHLOOLI  6 
For author affiliations, see end of text. 7 
Received November 14, 2007; Revised May 28, 2008; Accepted August 9, 2008 8 
This paper is available online at http://ijpt.iums.ac.ir 9 
ABSTRACT 10 
It is over several decades that ultrasound is used to enhance the transdermal drug delivery (phonphore-11 
sis). The mechanism of the enhancement is not fully understood and the ability of ultrasound on the en-12 
hancement for some drugs is unclear. The effect of continuous wave 870 KHz ultrasound at intensity of 1 13 
W/cm2 for 15 minutes on transdermal absorption of piroxicam from solution and gel formulations in hair-14 
less rat skin was studied. Exposure to ultrasound increased the rate of diffusion from gel and solution of 15 
piroxicam to 10 and 3 times higher than that in skins not exposed to ultrasound. We strongly believe that 16 
the lower diffusion of piroxicam from the solution is caused by extra-bubbles generated by ultrasound. It 17 
can be suggested that cavitation activity and its negative surface charges play a dominant rule in phono-18 
phoresis. 19 
Keywords: Piroxicam, Phonophoresis, Cavitation, Micro-streaming  20 
Transdermal drug delivery is designed to transfer 21 
drugs form the surface of the skin and through its vari-22 
ous layers into the systemic circulation [ 1]. In normal 23 
condition, the diffusion of drug into the skin and beyond 24 
that is limited. This limitation is attributed to stratum 25 
corneum of the skin. In order to increase diffusion of 26 
drugs through the skin, physical & chemical approaches 27 
have been adopted to change stratum corneum proper-28 
ties. Phonophoresis is a physical method to disturb the 29 
stratum corneum and is defined as the enhancement and 30 
movement of drugs through intact skin and into soft 31 
tissue under influence of ultrasound perturbation [ 2]. 32 
Although significant attention has been devoted to the 33 
investigation of phonophoresis, its mechanism of action 34 
has not been clearly understood. 35 
There are several parameters, which may affect the 36 
skin upon exposure of ultrasound. These parameters as 37 
summarised by Mitragotri are cavitation, thermal effect, 38 
induction/convection processes and mechanical effects. 39 
The role of cavitation in diffusion processes is well 40 
documented. Mitrogotri stated that enhancement of drug 41 
by phonophoresis is due to disordering of lipid bilayer 42 
of stratum corneum which is resulted from cavitation 43 
effects [ 3]. In another report, Mitragotri concluded that 44 
ultrasound has no effect on increasing diffusion of some 45 
drugs [ 4]. Some other researchers believed that the en-46 
hancement effect of phonophoresis was more pro-47 
nounced for polar compounds compared to non-polar 48 
compounds [ 5]. Although the importance of cavitation 49 
has been realised by other investigators, its effect on 50 
diffusion of drugs has not been fully described.  51 
Bubbles are usually produced in the sound field 52 
from small inactive bubble nuclei which are normally 53 
present in a medium activated by the pressure fluctua-54 
tion of the sound field. This causes volume pulsations of 55 
the nuclei, growth by rectified diffusion to resonant size, 56 
and concentration of acoustic energy in their vicinity 57 
[ 6, 7]. The nuclei will grow by a process of rectified dif-58 
fusion [ 7- 9]. Research by Watmough on cavitations 59 
revealed that the mapping of ultrasound field on paper is 60 
strongly dependent on the charge of the ions of the dyes 61 
and presence of micro-bubbles [ 10, 11]. Shiran has 62 
shown that there would be no pattern of ultrasound field 63 
in the absence of micro-bubble [ 12]. 64 
In this study, the effects of micro-bubbles and their 65 
electrical surface charges on the rate of drug diffusion in 66 
phonophoresis have been assessed. 67 
1735-2657/08/71-15-19 
IRANIAN JOURNAL OF PHARMACOLOGY & THERAPEUTICS 
Copyright © 2006 by Razi Institute for Drug Research (RIDR) 
IJPT 7:15-19, 2008 
16 |  IJPT  |  January 2008  |  vol. 7  |  no. 1  Shiran et al. 
 
MATERIALS AND METHOD 68 
A costume-made chamber with two compartments 69 
was   constructed   as shown in   Fig 1.   The    receiving  70 
compartment was covered with rubber (1 mm thickness) 71 
as an absorber to prevent standing wave formation and 72 
multiple reflections during sonification. The tempera-73 
ture of the skin inside receiving compartment was 74 
measured by a digital thermometer (LT Luton TM 905). 75 
Two plastic tubes (1 mm diameter) were fitted into the 76 
receiver compartment to take sample from, and to clear 77 
the air under the skin.  A magnetic stirrer with Perspex 78 
cover and speed of 100 rpm placed in a ban to keep 79 
temperature constant at 33oC throughout the experi-80 
ments. Male Wistar rats (200-300 g) were sacrificed by 81 
cervical dislocation and the abdominal hair was care-82 
fully removed with electrical clips. 83 
The stratum corneum side of the prepared abdominal 84 
skin was mounted on ring-shaped donor compartment 85 
with 2.5 cm diameter; large enough to allow the ultra-86 
sound to pass through without any disturbances. The 87 
donor and receiver compartments were screwed together 88 
and 55 ml of normal saline (9 g/L) was injected into the 89 
receiver compartment and left for 15 hours in refrigera-90 
tor for the skin to achieve steady state diffusion rate. 91 
Prior to the experiment, the skin was washed with 55 ml 92 
fresh normal saline. Pre-experimental solution was then 93 
replaced by fresh normal saline at 33ºC.  94 
A plane circular 870 KHz transducer, 4 cm in di-95 
ameter powered by sonostat 633 generator, was placed 96 
vertically in the donor compartment. The fixed skin in 97 
the chamber was covered either with 10 g of piroxicam 98 
gel (0.5%) or piroxicam solution (5×10 ml; 20 mg/ml) 99 
and exposed to ultrasound in continuous wave mode at 100 
intensity of 1W/cm2 for 15 minutes. The equipment was 101 
calibrated by radiation force and dye/paper method be-102 
fore use. Each experiment was repeated four times and 103 
the mean of the values were obtained. 104 
Aliquots of 5 ml were withdrawn from the receiver 105 
chamber immediately after the end of sonification and 106 
then at 2-hour intervals up to 6 hours. After withdrawal 107 
of each sample, the same volume of normal saline was 108 
added to keep the receiver chamber volume constant 109 
during experiment. The drug concentration was deter-110 
mined using HPTLC system. 111 
RESULTS 112 
Fig. 2 shows the results of continuous wave ultra-113 
sound applied to the skin covered with the piroxicam 114 
gel. The rate of piroxicam diffusion through the skin, 115 
using gel formulation, was over 10 fold higher com-116 
pared to its control for 6 hours of sampling, with sharp 117 
increase in piroxicam concentration two hours after the 118 
termination of sonification. 119 
The rate of diffusion of piroxicam from solution in 120 
 
Fig. 1. Two compartments chamber used for mounting the isolated skin 
 
Table 1. The mean value of the amount of piroxicam from gel formulation in the receiver chamber with and without ultrasound (US) radiation 
Amount of Piroxicam (μg)  
(Mean ± SE)  
Time 
(minute) Without (US) With (US) 
 
t-test 
Ratio of the amount of Piroxicam 
(with US/without US) 
15 0 0 - 00.0 
120 3.71±0.5 40.3±4.5 S* 10.9 
240 5.98±1.2 58.64±2.3 S 10.0 
360 6.55±1.2 76.41±3.9 S 11.6 
480 8.74±2.3 91.83±6.9 S 10.5 
*S=significant (p<0.05) 
Effect of Surface Charge on Phonophoresis ijpt.iums.ac.ir  | 17 
 
the presence and absence of ultrasound application is 121 
illustrated in Fig. 3. The graph shows an increase in 122 
piroxicam concentration in receiver chamber. The abso-123 
lute increase in concentration is about 3 times higher 124 
than its control with very low diffusion rate two hours 125 
after termination of sonication.  126 
The mean of the piroxicam concentrations found in 127 
receiver chamber with or without ultrasound radiation 128 
are shown in Tables 1 and 2 for gel and solution respec-129 
tively. The solution of piroxicam in the absence of ultra-130 
sound also shows a more linear penetration of the drug 131 
compared to the gel formulation. A maximum 7ºC tem-132 
perature increase was recorded for both sets of experi-133 
ments.  134 
DISCUSSION  135 
The results indicate that ultrasound has an enhancing 136 
effect on transdermal drug delivery. This enhancement 137 
is believed to be attributed to several factors such as 138 
thermal, cavitational and convective effects [ 3]. Ueda 139 
and co-workers found different diffusion rate for polar 140 
and non-polar drug, but they related this increase to 141 
thermal effect and diffusivity of the drug across the po-142 
lar region in the stratum corneum [ 5]. The temperature 143 
rise in this study was about 7°C, which according to 144 
previous studies can not have a great effect on the en-145 
hancement of piroxicam diffusion through the skin 146 
[ 2, 3]. 147 
The amount of piroxicam measured in receiver 148 
chamber from the gel formulation was almost 10 times 149 
higher than the control value, which was statistically 150 
significant. The concentration of piroxicam measured 151 
for the solution of piroxicam was almost three times 152 
higher compared to its control, even with higher con-153 
centration of drug applied to the skin. The increase of 154 
piroxicam permeation through the skin in both gel and 155 
solution forms upon ultrasound exposure signifies the 156 
involvement of other mechanisms rather than the 157 
change in the temperature alone. Since the increased 158 
permeation from gel is more pronounced than that from 159 
solution and also the possibility of bubble formation in 160 
solution is more than that in the gel, the bubble activi-161 
ties should be reviewed.  162 
It seems that cavitation was an important factor 163 
which caused the rate of diffusion of piroxicam from 164 
solution to be less than that from the gel despite the 165 
higher concentration of drug in the solution. Usually, 166 
there are some bubble nucleus existing in the liquid me-167 
dium and these nuclei will grow under rectified diffu-168 
Time (min)
0 100 200 300 400 500 600
A
m
ou
nt
 o
f p
iro
xi
ca
m
 ( μ
g)
0
20
40
60
80
100
120
-  Without US
z-  With US
 
  
 
Fig. 2. The time-concentration curve of piroxicam gel 
upon 15 minutes ultrasound treatment (data are means 
+ standard error) 
 Fig 3. The time-concentration curve of piroxicam solu-
tion upon 15 minutes ultrasound treatment (data are 
means + standard error) 
 
Table 2. The mean value of the amount of piroxicam in solution form in the receiver chamber with and without ultrasound (US) radiation 
Amount of Piroxicam (μg) 
(Mean ± SE)  Time 
(minute) Without (US) With (US) 
 
 
t-test 
Ratio of the amount of Piroxicam 
 
 (with US/without US) 
15 0 0 - 0.0 
120 12.3±4.3 22.43±5.9 NS* 1.8 
240 17.16±7.6 53.16±10 S** 3.1 
360 29.52±8.2 74.47±6 S 2.5 
480 41.24±9.8 79.45±4.5 S 2.0 
*NS= no significant, **S=significant (p<0.05) 
18 |  IJPT  |  January 2008  |  vol. 7  |  no. 1  Shiran et al. 
 
sion and form bubble in a sound field. There are three 169 
possible areas in which bubbles may form: in the liquid 170 
medium, on the surface of the skin and inside the epi-171 
dermis. Close examination of the surface of the skin 172 
shows that the surface of the skin contains a large num-173 
ber of holes about 0.4mm in diameter [ 13]. These holes 174 
accommodate gas nuclei and when sonicated, the air 175 
will expand forming gas bubbles and because of the 176 
temperature rise in the skin, rectified diffusion may also 177 
contribute to their growth, as shown in Fig. 4. Mitragotri 178 
et al. and Wu et al. strongly believed that the growth of 179 
bubble by rectified diffusion does take place in the 180 
keratinocytes and lipid bilayer [ 3, 14]. It was also be-181 
lieved that the presence of cavitation is the possible 182 
mechanism of the passage of high molecular weight 183 
drugs through the skin [ 14]. They realized that the cavi-184 
tation influenced the penetration of drug through the 185 
skin, but the mechanism of action was not completely 186 
explained. It was reported by a number of researchers 187 
that the gas bubbles possessed a considerable negative 188 
electrical charges on the surface [ 10, 15- 20]. Margulis 189 
and Shiran experimentally measured the charge on bub-190 
ble surface in distilled water to be 3×10-13 C and 10 ×10-191 
13 C respectively [ 19, 20]. Watmough et al. suggested 192 
that the charges on acoustically-excited bubbles are 193 
greater than gas/water interface in the absence of ultra-194 
sound [ 10]. They also stated that ultrasound is capable 195 
of maintaining the surface charges on the bubbles even 196 
in the presence of ions which would otherwise neutral-197 
ize the charge.  198 
The charged bubbles would thus be expected to at-199 
tract the cationic drug ions and thus increase the local 200 
concentration of drug. Anionic drug ions, on the other 201 
hand, would be repelled by the electric field set up by 202 
micro-bubbles. Since piroxicam ions are negatively 203 
charged, local stirring caused by micro-streaming in-204 
creases this depletion of piroxicam when resonant bub-205 
bles are excited by high local intensity [ 21]. Where the 206 
piroxicam concentration is maintained at its lowest 207 
level, less piroxicam enters the skin. There are greater 208 
numbers of induced gas bubble in piroxicam solution 209 
compared to gel formulation, which can repel the nega-210 
tively-charged piroxicam ions. This explains why the 211 
amount of piroxicam measured from sonicated solution 212 
of piroxicam with five times higher concentration was 213 
lower than that from the gel. Such large difference has 214 
not been observed in the control groups. In fact, the 215 
penetration of drugs through the skin in control experi-216 
ments with piroxicam solution was slightly higher. 217 
Bommannan et al. reported that penetration rate of drug 218 
through the skin for 2 MHz ultrasound was less than 219 
that for 16 MHz ultrasound [ 2]. They also stated that the 220 
treatment with ultrasound at 10 and 16 MHz signifi-221 
cantly increased penetration of drug (almost 5 fold). 222 
They could not observe significant difference between 223 
the ultrasound-treated and controls at these frequencies. 224 
They concluded that “the enhancing effect of sonopho-225 
resis is due to direct effect of ultrasound on, presuma-226 
bly, stratum corneum”. According to Hueter and Bolt 227 
“above 10 MHz frequency, a vapour type of cavitation 228 
is unlikely to occur” [ 22]. The presence of cavitation 229 
and presumably the negative charges of the drug ions 230 
suppressed the rate of penetration at 2 MHz frequency, 231 
while there were no bubbles at frequency of 10 and 16 232 
MHz. The evidence provided here also suggests that it 233 
took almost 2 hours before the bubble (on the surface 234 
and possibly inside the keratinocytes and lipid bilayer) 235 
could dissolve and the passage for piroxicam diffusion 236 
be cleared to accelerate the drug passage. 237 
In conclusion, this study has demonstrated that ultra-238 
sound significantly enhanced transdermal absorption of 239 
piroxicam from solution and gel formulations. It also 240 
suggests that the cavitation and its surface charge play a 241 
major rule in this enhancement. We also believe that the 242 
size of bubble over the surface of the skin, keratinocytes 243 
and lipid bilayer does affect the rate of permeation.  244 
 
Fig. 4. Formation of bubble on the surface of the skin during sonication in solution 
 
Effect of Surface Charge on Phonophoresis ijpt.iums.ac.ir  | 19 
 
REFERENCES 245 
1. Ranada VV. Drug delivery systems. Transdermal drug delivery 246 
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nistic study of ultrasonically-enhanced transdermal drug deliv-252 
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variation of the sonophoresis transdermal transport enhancement 255 
from drug to drug. J Pharmaceut Sci 1997; 86: 1190. 256 
5. Ueda H, Ogihara M, Sugibrayashi K, Morimoto Y. Difference in 257 
the enhancing effects of ultrasound on the skin permeation of 258 
polar and non-polar drugs. Chem. Pharm Bull 1996; 44: 1973-6. 259 
6. Minneart M. On musical air bubble and the sounds of running 260 
water. Phil Mag 1933; 16: 235-48. 261 
7. Flynn HG. Physics of acoustics cavitation in liquids. Physical 262 
Acoustics Vol. 1B, 1964; Ed. W. P. Mason, Academic Press, 263 
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Acoustic Bulletin 1992; March/April: 5-8. 270 
11.  Watmough DJ, Shiran MB, Quan KM, et al. Evidance that 271 
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the Sarvazyan method for recording ultrasound field distribu-275 
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Chem Soc 1939; 61: 2392-8. 288 
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31: 54-64 299 
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CURRENT AUTHOR ADDRESSES 303 
Mohammad Bagher Shiran, Department of Bio-Medical Physics, Iran 304 
Univ. of Med. Sci., Tehran, Iran. E-mail: Shiran@iums.ac.ir (Cor-305 
responding author)  306 
Manijeh Motevalian, Razi Institute for Drug Research (RIDR), Iran 307 
Univ. of Med. Sci., Tehran, Iran. 308 
Rezvan Ravanfar, Department of Bio-Medical Physics, Iran Univ. of 309 
Med. Sci., Tehran, Iran. 310 
Shahab Bohlooli, Department of Pharmacology, Iran Univ. of Med. 311 
Sci., Tehran, Iran. 312 
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The effect of bubble surface charge on phonophoresis: Implication in transdermal piroxicam delivery

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The effect of bubble surface charge on phonophoresis: Implication in transdermal piroxicam delivery

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