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Efficacy of Transdermal Magnesium Ascorbyl Phosphate Delivery after
Ultrasound Treatment with Microbubbles in Gel-Type Surrounding Medium
in Mice
Ai-Ho Liao, Ying-Jui Lu, Chi-Ray Hung
PII:
DOI:
Reference:
S0928-4931(15)30673-1
doi: 10.1016/j.msec.2015.12.058
MSC 6051
To appear in:
Received date:
Revised date:
Accepted date:
13 April 2015
7 July 2015
28 December 2015
Please cite this article as: Ai-Ho Liao, Ying-Jui Lu, Chi-Ray Hung, Ecacy of Transdermal Magnesium Ascorbyl Phosphate Delivery after Ultrasound Treatment with Microbubbles in Gel-Type Surrounding Medium in Mice, Materials Science & Engineering C
(2015), doi: 10.1016/j.msec.2015.12.058
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Efficacy of Transdermal Magnesium Ascorbyl Phosphate Delivery
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+886-2-27303742
Fax:
+886-2-27303742
E-mail: aiho@mail.ntust.edu.tw
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ABSTRACT
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increasing their viscosity through the addition of thickening agents. The present study
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first assessed the usefulness of ultrasound (US) plus US contrast agent, microbubbles
(MBs), in agarose gel for enhancing transdermal drug delivery. The effect of US plus
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MBs in agarose gel on the penetration of the skin by magnesium ascorbyl phosphate
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(MAP) was explored both in vitro and in vivo. In the in vitro experiments, the
stability of MBs was investigated by examining the penetration of MAP by the model
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drug, Evans blue, in two media: an agarose phantom and pig skin. The penetration
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depth in the agarose phantom and pig skin increased by 40% and 195%, respectively,
when treated with US plus MBs in 0.1% agarose solution combined with MAP
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(UMB1), and by 48% and 206%, respectively, when treated with US plus MBs in
0.15% agarose solution and MAP (UMB2). The skin-whitening effects in C57BL/6J
mice in the UMB1 and UMB2 groups over a 4-week experimental period were
significantly increased by 63% and 70%, respectively, in the fourth week. The
findings of this study suggest that the survival of MBs with US is affected by the
viscosity of the surrounding medium, and that in mice, treatment with US plus MBs in
a suitable agarose gel can increase skin permeability and enhance transdermal MAP
delivery.
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Keywords:
Microbubbles,
Agarose
gel,
Ultrasound,
Transdermal
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delivery,
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1. INTRODUCTION
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cosmetics solutions were applied on the animal skin after all visible bubbles had
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disappeared, but it was found that liquid microemulsions were more appropriate for
topical application. MB behavior in an US field was recently investigated through
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the attenuation of US propagation through the MB mixture [2]. Our previous study
demonstrates the penetration depth, concentration, and efficiency of transdermal
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-arbutin delivery during 4 weeks after US treatment with MBs solution in mice [3].
The present study explored the feasibility of a gel-type MB compound combined with
US for enhancing the penetration of transdermal magnesium ascorbyl phosphate
(MAP) delivery in vivo.
US-achieved sonophoresis is known to increase skin permeability, but the
fundamental mechanism underlying this effect is still unclear [4]. Shock waves and
microjets generated during inertial cavitations are thought to be responsible for
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transdermal permeability enhancement, with microjets exerting a significantly greater
effect than shock waves [5]. Such cavitation occurs at various sites, such as the
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coupling medium, the skin surface, and the skin tissue [4]. The viscosity, surface
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tension, density, acoustic impedance, and other bulk and interfacial properties of the
coupling medium play an important role in the enhancement of skin permeability [6].
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The most common type of drug delivered through the skin using high-frequency
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ketorolac, and piroxicam [6]. Moreover, oral NSAIDs generally cause gastrointestinal
side effects, including nausea, heartburn, gastrointestinal ulcers, and nonspecific
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colitis [7]. Therefore, the combination of topical NSAID therapy and US is promising;
however, the combination of gel-type MBs plus US for TDD is necessary for such
therapy.
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impractical because it is readily soluble in water and is extremely unstable [9].
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Shaikh and Mashood (2014) determined the effectiveness of treating melasma with a
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combination of topical 5% MAP and pulsed fluorescent light, and found that
combining 5% MAP with pulsed fluorescent light is an effective, well-tolerated, and
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enhancement of skin permeability has been demonstrated. The properties of liquid and
gel-type MBs used for US enhanced transdermal MAP delivery were demonstrated
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both in vitro and in vivo. US combined with gel-type MBs was found to induce
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previous studies [17, 18]. Briefly, albumin-shelled MBs were generated by sonication
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and perfluorocarbon gas in physiological saline (pH 7.4, 0.9% sodium chloride) using
a sonicator (Branson Ultrasonics, Danbury, CT, USA) for 2 min. The number of
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(meanSD) for a size range of 0.82 m, and their mean diameter was 1195 nm.
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Two amounts of agarose powder (0.1 and 0.15 mg; FB-HA0604, FocusBio,
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Shaker RS-01, TKS, New Taipei City, Taiwan) for 20 min. The agarose solution was
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then placed into the sonication tank (Delta ultrasonic cleaner d150h, Delta, Hsin-Chu
City, Taiwan) to remove the excess gas, and finally stored at 4C in a refrigerator.
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Before the experiments were performed, the gel-type MBs were prepared in either
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filtered with a 5 m syringe filter (Critical Process Filtration, Inc., Nashua, NH, USA)
and then hardened by 0.002% glutaraldehyde (Nippon Shiyaku Co., Tokyo, Japan).
The morphology of the hardened albumin MBs was studied using scanning electron
microscopy (SEM) (Quanta 3D FEG, FEI, ORR, USA). The samples were prepared
for SEM by coating with platinum. SEM images were recorded on a system at an
accelerating voltage of 30 kV.
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agarose gel during sonication using a US animal-imaging system (Prospect, S-Sharp
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enhancement of the penetration depth was greatest for 2-W/cm2 US and the condition
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was used either for the in vitro skin penetration or for the animal treatments in this
study [3]. During the US imaging, the loaded phantom was sonicated using the
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NepaGene, Ichikawa, Japan) at an acoustic intensity of 2 W/cm2 for 1 min. The duty
cycle was set at 50%, the PRF was set at 250 msec and a 6-mm-diameter transducer
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was used. The region of interest was drawn over the entire MB-loaded chamber in a
two-dimensional imaging plane by an operator, the dynamic range was set at 50 dB
and the average pre- and postsonication image intensities were measured in B-mode
images.
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phantoms were constructed with a diameter of 1.2 cm and a height of 5 mm (encircled
with US gel to prevent leakage); the round area of each phantom was loaded with
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Evans blue or MBs. The probe of the sonoporation gene transfection system (ST
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2000V, NepaGene) was placed 5 mm from the top of the phantom. After adding
500 l of the 0.1% agarose gel, 0.15% agarose gel, or the MBs at four different
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0.15% agarose gel, the area was sonicated using the 1-MHz US transducer of the
sonoporation system at an acoustic intensity of 2 W/cm2 for 1 min. The duty cycle
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was set at 50% and a 1.2-cm-diameter transducer was used. The change in
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surface and the area was washed three times for 1 min with physiological saline. The
Evans-blue solution was then injected into the same area on the phantom and the
sample left for 5 min to allow penetration. The Evans blue was then removed and the
area was washed three times for 1 min with physiological saline. Sections of the
phantom were cut at a thickness of 2 mm and prepared for light-microscopy
evaluation.
The penetration depths of the Evans blue were measured using MATLAB (The
MathWorks, Natick, MA, USA). At first, the light-microscopy images were converted
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into grayscale images and image histogram-based binarization was performed [19].
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performed based on the histogram for many experiments [19]. Then, the boundary
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was detected by Sobel-operator-based edge detection [20]. At this step, the same
threshold (100-230) was used when processing all of the images. Finally, the area of
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the penetration region was measured and the area was divided by the length of the
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x-axis of the image to get the mean penetration depth (y-axis) of the Evans blue.
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Fresh porcine skin was obtained from a local slaughterhouse. The protocol for
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the experiments involving pig skin was similar to that described for the agarose
phantom. The treatment area on the 2-mm-thick pig skin was sonicated by the 1-MHz
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embedded samples were placed on the 25C freezing stage of a cryostat (Microm
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microscopy slides, air-dried at room temperature, and then mounted for microscopy
examination. The average penetration depth was measured by sampling 10 depths on
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each slide (15 slices per experiment and each experiment repeated 4 times).
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model [21]. Five-week-old mice weighing 2025 g were obtained from Bio Lasco
(Taipei, Taiwan). The experimental protocol was approved by the Institutional Animal
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Care and Use Committee of the National Taiwan University Hospital, Taipei, Taiwan.
Animal care complied with institutional guidelines and regulations. Throughout the
experiments, the animals were housed in stainless-steel cages in an air-conditioned
room with the temperature maintained at 2528C and with alternating light and dark
cycles lasting 12 hours each. The animals were acclimatized for 7 days prior to the
experiment. After their hair had been removed, the skin color was measured using a
Chroma Meter (CR-400, Konica Minolta Sensing, Tokyo, Japan). The animals were
exposed to ultraviolet radiation B (UVB) irradiation (G8T5E, Sankyo, Tokyo, Japan)
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to
induce
hyperpigmentation
(total
energy
dose
per
exposure=1 J/cm2,
wavelength=306 nm, three times/week for 2 weeks), and then the skin color was
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measured again.
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The details of the experimental design are shown in Fig. 1. The animals were
divided into the following five groups (n=5 per group, treatment applied three
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times/week for 4 weeks): (1) control (no treatment, C), (2) penetrating MAP only (M),
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(3) US combined with penetrating MAP (UM), (4) US plus MB contrast agent in
0.1% agarose solution combined with penetrating MAP (UMB1), and (5) US plus MB
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contrast agent in 0.15% agarose solution combined with penetrating MAP (UMB2).
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The change in skin color induced by each of the treatments was assessed at
predetermined times using the Chroma Meter. The luminosity index, L [22], was
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2.7 Histochemistry
Skin tissue samples (approximately 88 mm2) were taken from the treatment
area immediately after the experiments and stored in 10% formalin solution.
Hematoxylin and eosin (Sigma-Aldrich) staining was applied, and the samples were
analyzed to evaluate the relative melanin content and damages in the skin structure
after US treatments.
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2.8 Statistical analysis
The obtained data were analyzed statistically using Students t-test. A probability
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3. RESULTS
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area of the MBs surface in agarose was found for gels generated by copolymerization
(Fig. 2(B), (C)). US images of 4107 bubbles/ml albumin-shelled MBs, MBs in 0.1%
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agarose gel, MBs in 0.15% agarose gel without and with US sonication at 2 W/cm2
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for 1 min are shown in Fig. 3(A)(C) and 3(D)(F), respectively; the signal intensities
in panels Fig. 3(A)(F) are quantified in Fig. 3(G). The image intensities of the MBs
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at
various
concentrations
(0.23107,
4.6107,
11.5107,
and
23107 bubbles/ml). Both the agarose concentration and the concentration of MBs
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affected the penetration depth. At the MBs concentration of 0.23107 bubbles/ml, the
penetration depth was enhanced significantly more when using 0.1% agarose gel than
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penetration depth was enhanced significantly more when using 0.15% agarose gel
than 0.1% agarose (p<0.05). The penetration depth was enhanced significantly more
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when using 0.1% agarose gel combined with 23107 bubbles/ml and 0.15% agarose
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gel combined with 4.6107 bubbles/ml than their control group (p<0.001). Figure 5
[(A)(D) and (E)(H)] shows microscopy images before and after imaging processing
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of the agarose phantoms without US, with US (at an acoustic intensity of 2 W/cm2),
US combined with MBs (23107 bubbles/ml) in 0.1% agarose, and US combined with
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MBs (4.6107 bubbles/ml) in 0.15% agarose when the Evans-blue solution was
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allowed to stand for 5 min. In the US-only group there was no significant difference
in the penetration depth (p>0.05) compared to the control condition. However, the
penetration depths of MBs (23107 bubbles/ml) in 0.1% agarose, and US combined
with MBs (4.6107 bubbles/ml) in 0.15% agarose were 44% and 44.7% greater than
in the control
group
(p<0.01).
greater for
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3.3 Evaluation of the penetration depth in pig skin
The pig-skin samples in groups UM (US plus water, 0.1% agarose gel or 0.15%
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agarose gel, combined with Evans blue), UMB (US plus MB contrast agent in water
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combined with Evans blue), UMB1 (US plus MB contrast agent in 0.1% agarose gel
combined with Evans blue), and UMB2 (US plus MB contrast agent in 0.15% agarose
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gel combined with Evans blue) were cryosectioned for light-microscopy evaluation at
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concentrations of MBs and agarose. The degree of penetration in either the cuticle or
the epidermis was significantly greater in groups UMB, UMB1, and UMB2 than in
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MBs in water was significantly greater than in agarose gel (p<0.05). For MB contrast
agent in agarose gel, the greatest conditions were 11.5107 bubbles/ml MBs in 0.1%
agarose (14.81.1 m) and in 4.6107 bubbles/ml MBs in 0.15% agarose
(14.40.8 m). The greatest conditions were not significantly different from 4.6107
MBs in water (p>0.05). The penetration depths in those groups treated with 0.1% and
0.15%
agarose
were
5.00.6
and
4.60.6 m,
respectively.
When
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US only in 0.1% and 0.15% agarose, respectively (p<0.05). Therefore, the
enhancement of the penetration depth was dependent upon the proper proportions of
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MBs and agarose gel for both the in vitro skin penetration and for the animal
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Figure 8 shows photographs of the mouse skin (Fig. 8AE) and histology images
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(Fig. 8FJ) for groups C (Fig. 8A, F), M (Fig. 8B, G), UM (Fig. 8C, H), UMB1
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(Fig. 8D, I), and UMB2 (Fig. 8E, J) at week 4. Figure 9 plots the brightness values
(i.e., L) to demonstrate the whitening effects of MAP, US, and gel-type MBs on
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UV-induced hyperpigmentation over a 4-week period. Fig. 8B, Fig. 8C and Fig. 9
show that the brightness of the skin in groups M and UM had recovered. The skin
brightness was more effectively increased and closer to the original color in groups
UMB1 and UMB2 than in groups C, M, and UM. The skin brightness in the
observation area was more uniform in groups UMB1 and UMB2 than in groups C, M,
and UM. Moreover, no skin damage was evident in any of the US treatment groups.
Histology analysis revealed that both the UMB1 and UMB2 treatments resulted in a
significant decrease in the relative melanin content of the skin. No changes in the skin
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structure or bilayerbilayer interface were observed in any of the treatment groups.
In Fig. 9, the brightness value was around 36.238.4 in each group after UVB
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exposure. At week 1 the brightness values in groups UM, UMB1, and UMB2 had
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increased by 25%, 31%, and 31%, respectively. Significant differences (p<0.05) were
obvious between each US treatment group and non-US treatment group (i.e., C and
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M), but not between groups UM, UMB1, and UMB2 (p>0.05). At week 2 the
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brightness values in groups M, UM, UMB1, and UMB2 had increased by 35%, 41.8%,
49.6%, and 57.7%, respectively. At week 3 the brightness values in groups UMB1 and
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UMB2 had increased by 58.6% and 63.1%, respectively, making them close to the
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original skin color, while those in groups M and UM had increased by smaller
amounts (by 52.1% and 54.5%, respectively). At week 4 the brightness values in
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groups M, UM, UMB1, and UMB2 had reached a plateau, with increases of 57%,
61.4%, 63.3%, and 69.9%, respectively, while that in group C had increased by
31.1%.
4. DISCUSSION
Gel cream containing vitamin C can be applied to the skin in patients to suppress
melanin formation by tyrosinase and melanoma cells therein [23]. Since a solution
containing MBs has a very low viscosity, it cannot be applied directly to the skin. The
usual way of resolving this problem is to add a suitable thickening agent that can
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modify the rheological properties without significantly influencing the other features
of MBs [24, 25]. In the present study, MBs in agarose gel were applied with US to the
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skin to enhance MAP delivery. Agar gels have been found useful in the fields of
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medicine and pharmacy [26], and are commonly used in the food industry as
thickening and gelling agents [26, 27]. Moreover, agarose gel with graphite suspended
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tissue (e.g., speed of sound and average attenuation) [28]. Rozman and Gasperlin
(2007) used gel-like microemulsions as carrier systems for the dermal delivery of
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vitamins C and E, and found that these microemulsions offered the best protection for
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both vitamins and increased their stability compared with that of a basic vitamin
solution [29]. Those authors also found that the addition of thickener significantly
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increased the viscosity and changed the behavior of the delivery systems. According
to the results of the present study, although agarose gel did not affect the survival of
MBs before sonication, it did affect their behaviors with US treatments and the
efficacy of drug penetration. This efficacy can be controlled by optimizing the MB
conditions, such as their concentration. According to the results of phantom study
(Fig. 5), the use of optimum concentrations of agarose and MBs0.1% agarose gel
combined with 23107 bubbles/ml and 0.15% agarose gel combined with
4.6107 bubbles/mlsignificantly enhanced the MAP penetration depth. In pig skin,
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although the penetration of 23107, 11.5107, and 0.23107 MB in water was
significantly greater than in agarose gel. It is not easy to encircle with US gel to
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prevent leakage and apply to the skin surface in living animals. However, MBs in gel
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combined with 11.5107 bubbles/ml and 0.15% agarose gel combined with
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4.6107 bubbles/ml. These two conditions were not significantly different from
4.6107 MBs in water. Moreover, the penetration in pig skin with 0.15% agarose gel
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was more uniform than with 0.1% agarose gel at all concentrations of MBs tested.
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Thus, 0.15% agarose gel could improve the uniformity of the effects of MBs and US
on the skin surface. This finding is consistent with those of the small-animal
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experiments, which demonstrated that the skin brightness in the observation area was
more uniform in groups UMB1 and UMB2 than in the other groups.
In animal experiments, the skin brightness values of the control group (C)
increased very slowly, and those of the MAP group (M) increased relatively slowly in
the first week. However, during the same period, the skin brightness values of the UM,
UMB1, and UMB2 groups increased more clearly. By the second week the rate of
brightness increase in the M group was similar to that in the UM, UMB1, and UMB2
groups. During the 4 weeks of observation, the brightness values of the UMB1 and
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UMB2 groups were similar, and higher than those of the UM group. This may be due
to the cavitation induced by US, which may have improved the permeability of the
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lipid bilayer membrane of the stratum corneum for a short time. Thus, combined
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gel-type MBs and US and US alone can enhance MAP delivery at the early stage.
Combining US with MBs increased the delivered concentration of MAP and did not
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influence the time trend of MAP delivery. After US treatment with gel-type MBs in
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mice, more MAP passed through the stratum corneum lipid bilayer and directly
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5. CONCLUSIONS
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concentrations were determined. The in vivo results demonstrated that combination
treatment with US and MBs in agarose gel (at optimum concentrations of both) can
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ACKNOWLEDGMENTS
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increase skin permeability in mice and thus enhance MAP delivery to inhibit
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This work was supported by the Ministry of Science and Technology of the
Republic of China, Taiwan under Contract of MOST 100-2628-E-011-015-MY3 and
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Measurements of lifetime and attenuation properties of ultrasound/magnetic
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of the H/L and L/H microemulsions, STP Pharma Sci 10 (2000) 220223.
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FIGURE CAPTIONS
Fig. 1. Experimental design and protocol. The skin color was measured before and
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after the animals were exposed to UVB irradiation. The change in skin color induced
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Fig. 2. SEM micrograph of the (A) MBs , (B) MBs in 0.1% agarose, and (C) MBs in
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0.15% agarose.
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for 1 min. The signal intensities in panels AF are quantified in panel G. The signal
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intensity of MBs decreased more significantly in 0.1% and 0.15% agarose than in
physiological saline after sonication (*p0.001, **p0.0001). Data in panel G are
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at
various
concentrations
(0.23107,
4.6107,
11.5107,
and
23107 bubbles/ml). 0.1% agarose gel combined with 23 107 bubbles/ml and 0.15%
agarose gel combined with 4.6 107 bubbles/ml enhanced the penetration depth
significantly than other groups (*p0.05, **p0.001).
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Fig. 5. Light-microscopy images before (AD) and after (EH) imaging processing of
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combined with MBs (4.6107 bubbles/ml) in 0.15% agarose. The penetration depths
of Evans blue are quantified in panel I. The penetration depth was greater for
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23107/ml MBs in 0.1% agarose and 4.6107/ml MBs in 0.15% agarose than in the
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UMB1 (middle row), and UMB2 (bottom row) for MBs at various concentrations.
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Fig. 7. Quantification of the penetration depths of Evans blue shown in Fig. 5. The
penetration of 23107, 11.5107, and 0.23107 MBs in water was significantly greater
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than in agarose gel (p<0.05). The degree of penetration was greatest with
11.5107 bubbles/ml MBs in 0.1% agarose (14.81.1 m) and in 4.6107 bubbles/ml
MBs in 0.15% agarose (14.40.8 m) (*p0.05). These conditions were not
significantly different from 4.6107 MBs in water (**p>0.05).
Fig. 8. Photographs (AE) and histology images (FJ) of mouse skin from groups C,
M, UM, UMB1, and UMB2 at week 4 of treatment.
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Graphical abstract
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Highlights
1. the survival of MBs with US is affected by the viscosity of the surrounding medium
2. treatment with US plus MBs in an optimum agarose gel increases skin permeability
3. skin penetration of some concentrations of MBs in water was greater than in gel
4. MBs in gel is more easy to apply to skin surface in living animal
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