RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Erbium–Yttrium–Aluminum–Garnet Laser Irradiation Ameliorates Skin Permeation and Follicular Delivery of Antialopecia Drugs WOAN-RUOH LEE,1,2 SHING-CHUAN SHEN,1 IBRAHIM A. ALJUFFALI,3 YI-CHING LI,4,5 JIA-YOU FANG4,6,7 1
Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan Department of Dermatology, Taipei Medical University Shuang Ho Hospital, New Taipei City, Taiwan 3 Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 4 Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan 5 Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan 6 Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan 7 Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan 2
Received 21 April 2014; revised 24 July 2014; accepted 6 August 2014 Published online 3 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24143 ABSTRACT: Alopecia usually cannot be cured because of the available drug therapy being unsatisfactory. To improve the efficiency of treatment, erbium–yttrium–aluminum–garnet (Er–YAG) laser treatment was conducted to facilitate skin permeation of antialopecia drugs such as minoxidil (MXD), diphencyprone (DPCP), and peptide. In vitro and in vivo percutaneous absorption experiments were carried out by using nude mouse skin and porcine skin as permeation barriers. Fluorescence and confocal microscopies were used to visualize distribution of permeants within the skin. Laser ablation at a depth of 6 and 10 m enhanced MXD skin accumulation twofold to ninefold depending on the skin barriers selected. DPCP absorption showed less enhancement by laser irradiation as compared with MXD. An ablation depth of 10 m could increase the peptide flux from zero to 4.99 and 0.33 g cm−2 h−1 for nude mouse skin and porcine skin, respectively. The laser treatment also promoted drug uptake in the hair follicles, with DPCP demonstrating the greatest enhancement (sixfold compared with the control). The imaging of skin examined by microscopies provided evidence of follicular and intercellular delivery assisted by the Er–YAG laser. Besides the ablative effect of removing the stratum corneum, the laser may interact with sebum to break up the barrier function, increasing the skin delivery of antialopecia drugs. The minimally invasive, well-controlled approach of laser-mediated drug permeation offers a potential way to treat alopecia. This study’s findings provide the basis for the first report on laser-assisted delivery C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3542–3552, 2014 of antialopecia drugs. Keywords: alopecia; minoxidil; diphencyprone; peptide; laser; absorption; percutaneous; skin; macromolecular drug delivery
INTRODUCTION Alopecia is known as hair loss on the scalp because of an androgenetic process (androgenetic alopecia) or an inflammatory process (alopecia areata). The prevalence of androgenetic alopecia in Caucasian men is 96%.1 On the contrary, alopecia areata affects 2.1% of the population.2 Minoxidil (MXD) is developed for the treatment of both alopecia types. This drug at a 5% dose is the first-line topical therapy for androgenetic alopecia.3 It can reduce baldness by the mechanisms of vasodilation, enhanced proliferation, and angiogenesis. Diphencyprone (DPCP) is a contact allergen for topical immunotherapy of alopecia areata. Alopecia treatment by drugs is always inefficient. The effect of MXD is not permanent, and the treatment cessation contributes to hair loss in 4–6 months.4 Moreover, contact dermatitis occurs in 6% of patients receiving 5% MXD.5 A lag time of 3 months from initiation of treatment to initial hair growth is observed for DPCP with a success rate of only 50%. The adverse effects of DPCP occur in 24% of patients.6 Recent advances in biotechnology have developed macromolecules and nanoparticles for alopecia therapy. These Correspondence to: Jia-You Fang (Telephone: +886-3-2118800; Fax: +886-32118236; E-mail:
[email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 3542–3552 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
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include growth factors, proteins, genes, and fullerenes.7–9 Abundant investigations document the use of peptides to counter the effect of alopecia. These include soymetide-4,10 formyl– methyonyl–leucyl–phenylalanine,11 prolactin,12 and calcitonin gene-related peptide.13 These biologics, however, show unsatisfactory results in treating alopecia because of their instability and molecular size, which is too large to penetrate the skin. Efficient topical drug delivery is challenging because of the formidable barrier function of the stratum corneum (SC). SC removal by tape-stripping, mechanical abrasion, and laser treatment has shown to be effective for promoting drug permeation via the skin.14 The approaches of tape-stripping and abrasion are limited and lack reproducibility because of the poor ability to control the SC ablation level. Laser treatment can precisely and selectively remove SC in a controlled and noncontact manner.15 The duration of laser irradiation is in the range of nano- to microseconds, indicating a quick operation for enhancing drug absorption.16 It has been shown that the ablative lasers can increase topical delivery of small molecule drugs, macromolecules, and nanoparticles.17,18 The improvement of antialopecia drug delivery for achieving efficacious therapy is urgent. We sought to evaluate the impact of ablative laser treatment on cutaneous delivery of antialopecia drugs, including MXD, DPCP, and peptide. Hair follicles are the main target for these drugs in treating hair loss. The follicular openings of
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
the scalp occupy 10% of the total scalp area.19 Whether laser treatment could facilitate drug delivery into the follicles was examined in this report. Using the erbium–yttrium–aluminum–garnet (Er–YAG) laser as the ablative tool, we measured the in vitro and in vivo skin permeation of antialopecia drugs via nude mouse and porcine skins. The role of the follicles on laser-assisted drug delivery was evaluated using the cyanoacrylate casting technique. The biodistribution of permeants in skin tissues was imaged by fluorescence microscopy and confocal laser scanning microscopy (CLSM) for vertical and horizontal observation, respectively. In our study, we demonstrated for the first time the utilization of the Er–YAG laser for topical delivery of antialopecia actives.
MATERIALS AND METHODS Materials Minoxidil, DPCP, rhodamine B, Nile red, polyethylene glycol (PEG) 400, and propylene glycol (PG) were purchased from Sigma–Aldrich (St. Louis, Missouri). Superglue (ethyl cyanoacrylate 7004T) was obtained from 3M (Taipei, Taiwan). Fluorescein isothiocyanate (FITC)–Pro–Arg–Leu–Leu–Tyr– Ser–Trp–His–Arg–Ser–His–Arg–Ser–His–COOH was synthesized by Tools Biotechnology (New Taipei City, Taiwan). Animals Female nude mice (ICR-Foxn1nu), 8-week-old, were supplied by National Laboratory Animal Center (Taipei, Taiwan). Specific pathogen-free pigs, 1-week-old, were provided by Animal Technology Institute Taiwan (Miaoli, Taiwan). The animal experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University. All animals used in this work were treated under the institutional guidelines. Preparation of Skin In the in vitro experiment, full-thickness skin from the dorsal area of the nude mice and pigs was excised after sacrifice. The sebum-removal skin was prepared by washing the SC side of the skin using cold hexane (4◦ C) five times.20 Er–YAG Laser The Er–YAG laser (Contour; Sciton Laser, Palo Alto, California) irradiated a wavelength of 2940 nm with a pulse duration of 100 :s. A scanning hand piece was employed to ablate a skin area of 1.5 × 1.5 cm2 . The ablated depth of the skin by the laser was set to 6 and 10 :m by fluences of 1.5 and 2.5 J/cm2 , respectively. In Vitro Skin Permeation Skin penetration of antialopecia drugs was measured by a Franz diffusion cell. The nude mouse or porcine skin with or without laser exposure was mounted between the donor and receptor compartments. The donor vehicle was 0.5 mL 30% PEG400–water, 30% PG–water, and water for MXD, DPCP, and FITC–peptide, respectively. The dose of MXD, DPCP, and FITC–peptide was 0.2% (w/v), 0.08%, and 0.028%, respectively. The receptor medium consisted of 30% PG–pH 7.4 buffer, 30% ethanol–pH 7.4 buffer, and pH 7.4 buffer for these three permeants. The effective diffusion area between compartments was DOI 10.1002/jps.24143
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0.785 cm2 . The stirring rate and temperature were kept at 600 rpm and 37◦ C, respectively. At appropriate intervals, 300 :L of the receptor medium was taken and immediately replaced by an equal volume of fresh medium. The samples of MXD and DPCP were analyzed by HPLC as described previously.21 The samples of FITC–peptide were assayed by fluorescence spectrophotometry. The excitation and emission wavelengths were set to 490 and 520 nm, respectively. The accumulation of permeants within the skin was measured after a 24-h delivery. The skin was removed from the Franz cell, then rinsed with water and blotted with tissue paper. The skin sample was weighed and minced by scissors, positioned in a glass homogenizer with 1 mL methanol for samples treated by MXD and DPCP, and ground for 5 min with an electric stirrer. The homogenization medium for the peptide was 0.1 N HCl. The mixture was centrifuged at 9615 xg for 10 min. After filtration via the polyvinylidene fluoride (PVDF) membrane, the samples were detected by HPLC or fluorescence spectrophotometry. Hair Follicle Uptake Differential stripping and cyanoacrylate skin surface casting were used to detect the content of the permeants in the follicles.22 Subsequent to stripping the SC of the skin removed from the Franz cell, a follicular cast was prepared. A drop of superglue was added on a glass slide, which was pressed onto the surface of the SC-stripped skin. The cyanoacrylate polymerized, and the slide was expelled with one quick movement after 5 min. The superglue remaining on the slide was scraped off and positioned in a tube with 2 mL methanol. The tube was shaken for 3 h. The final product was vacuumed to evaporate methanol. Mobile phase or water was added to dissolve the residuals for an HPLC or fluorescence spectrophotometry assay. Vertical Observation of Skin by Fluorescence Microscopy This experiment was performed in an in vitro Franz cell model by using porcine skin as a permeation barrier. Rhodamine B (0.3%, w/v, in 30% PEG400/water), Nile red (0.01%, w/v, in 30% PG/water), or FITC–peptide (0.028%, w/v, in water) was applied to the donor for a 6-h delivery. After the termination of skin permeation, the skin samples were sectioned in a cryostat microtome at a thickness of 20 :m, and then mounted by glycerin and gelatin. The slices were monitored with an inverted fluorescence microscope (IX81; Olympus, Tokyo, Japan) using a filter set at 450–490 and 515–565 nm for excitation and emission, respectively. Hematoxylin and eosin (H&E) stained the skin sections for observing the skin structure under bright-field imaging. Horizontal Observation of Skin by CLSM This experiment was carried out using an in vivo nude mouse model. A glass cylinder with a hollow area of 0.785 cm2 was attached to the dorsal skin by superglue. An aliquot of 0.2 mL of vehicles, the same as used with the experiment of fluorescence microscopy, was pipetted into the cylinder. The application period was 3 h. The animal was then sacrificed, and the treated skin region was excised. The skin thickness was scanned at 5-:m increments via Z-axis of confocal microscopy (TCS SP2; Leica, Wetzlar, Germany). The excitation and emission wavelengths for rhodamine B and Nile red were set to 543 and 560–620 nm, respectively. The wavelengths of excitation and Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
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Table 1. The In Vitro Skin Accumulation (:g/g) and Flux (:g/cm2 /h) of MXD, DPCP, and Peptide via Nude Mouse Skin Treated with or Without Lasers
Drug MXD
DPCP
Peptide
Laser Depth (:m) 0 6 10 0 6 10 0 6 10
Skin Accumulation (:g/g) 16.47 40.14 106.82 115.39 117.55 137.18 1.15 1.59 2.24
± ± ± ± ± ± ± ± ±
3.88 10.07 16.66 20.06 35.28 26.84 0.61 0.40 0.49
ERSA – 2.44 6.49 – 1.02 1.19 – 1.38 1.95
Flux (:g cm−2 h−1 ) ± ± ± ± ± ± 0 0.51 ± 4.99 ±
1.45 4.36 17.75 7.35 7.21 9.29
0.34 1.10 2.24 0.79 2.45 1.23 0.08 0.98
ERflux – 3.01 12.24 – 0.98 1.26 – – –
ERSA , enhancement ratio of the skin accumulation of laser-treated group/the skin accumulation of untreated control group; ERflux , enhancement ratio of the flux of laser-treated group/the flux of untreated control group; –, not determined. Each value represents the mean and SD (n = 4).
Table 2. The In Vitro Skin Accumulation (:g/g) and Flux (:g/cm2 /h) of MXD, DPCP, and Peptide via Porcine Skin Treated with or Without Lasers
Drug MXD
DPCP
Peptide
Laser Depth (:m) 0 6 10 0 6 10 0 6 10
Skin Accumulation (:g/g)
ERSA
Flux (:g cm−2 h−1 )
ERflux
± ± ± ± ± ± ± ± ±
– 8.77 6.53 – 1.79 1.62 – 1.06 1.67
0.17 ± 0.05 0.58 ± 0.10 0.89 ± 0.44 1.51 ± 0.36 5.90 ± 1.76 5.72 ± 0.09 0 0.04 ± 0.01 0.33 ± 0.10
– 3.41 5.24 – 3.91 3.79 – – –
1.03 9.03 6.73 11.38 20.42 18.44 0.64 0.68 1.07
0.21 2.08 1.53 3.75 3.75 3.51 0.23 0.19 0.27
ERSA , enhancement ratio of the skin accumulation of laser-treated group/the skin accumulation of untreated control group; ERflux , enhancement ratio of the flux of laser-treated group/the flux of untreated control group; –, not determined. Each value represents the mean and SD (n = 4).
emission were set to 488 and 510–540 nm for the skin receiving FITC–peptide. Images were taken by summing 15 fragments at different depths from the skin surface. Statistical Analysis Statistical analysis of differences between the groups was carried out using the Kruskal–Wallis test. The post-hoc test used for checking individual differences was Dunn’s test. A 0.05 level of probability (p < 0.05) was taken as the level of significance.
RESULTS In Vitro Skin Permeation An in vitro percutaneous absorption was first performed to evaluate the effect of laser irradiation on skin transport of antialopecia drugs. Delivery of drugs into/across nude mouse skin and porcine skin is summarized in Tables 1 and 2. The drug retained in the skin reservoir dictates accumulation to the superficial skin layer, whereas the permeant received in the receptor compartment simulates the extent of diffusion Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
into a deeper skin strata or circulation in an in vivo condition. The cumulative amount of permeant in the receptor as a function of time was calculated as flux, which followed in zeroorder fashion. As shown in Table 1, laser treatment delivered significantly (p < 0.05) more MXD into the skin than the nontreated group. The enhancement effect of the laser is shown in Table 1 in terms of the enhancement ratio (ER). The laser demonstrated more than a twofold- and sixfold-greater skin accumulation of MXD via laser-treated skin at an ablation depth of 6 and 10 :m than intact skin. A similar trend was revealed with flux values. Laser treatment followed by DPCP application slightly increased skin accumulation and flux. However, this enhancement did not achieve a significant difference(p > 0.05) as compared with the control group. Laser ablation resulted in the enhancement of FITC–peptide accumulation within the skin, although the ER was lower than that from MXD. The increment in ablation depth from 6 to 10 :m led to a significant elevation in peptide skin deposition (ER: 1.4 vs. 2.0). The receptor amount of FITC–peptide by passive diffusion was below the detection limit during a 24-h application. Laser ablation of 6 and 10 :m to nude mouse skin resulted in an increase in FITC–peptide flux from 0 to 0.5 and 5.0 :g cm−2 h−1 , respectively. Table 2 shows drug permeation profiles of porcine skin. MXD deposition in the skin increased significantly (p < 0.05) from 1.0 to 9.0 :g/g by a 6-:m peeling. Increasing the ablation depth from 6 to 10 :m did not increase the skin accumulation (p > 0.05). MXD flux with laser treatment at 6 and 10 :m was superior (p < 0.05) to that of the nontreated group with ER of 3.4 and 5.2, respectively. In the porcine skin model, the laser could improve the skin accumulation and the flux of DPCP (p < 0.05). An increment of about 20-fold in DPCP skin deposition was found with laser treatment at both 6 and 10 :m. DPCP flux by laser resurfacing at 6 and 10 :m was fourfold higher than that of the nontreated control. With respect to FITC–peptide, an ablation depth of 6 :m was insufficient to enhance skin uptake (p > 0.05). Skin accumulation of FITC–peptide by laser exposure at a 10-:m depth was 1.7 times greater than that of intact skin (p < 0.05). An increase of the ablation depth from 6 to 10 :m led to a further enhancement of FITC–peptide flux from 0.04 to 0.33 :g cm−2 h−1 . Hair Follicle Uptake It is important to increase the uptake of antialopecia drugs into the hair follicles. A differential stripping technique was used to detect the drug amount selectively accumulated in the follicles as depicted in Figure 1. The recovery of MXD from casts of intact nude mouse skin and porcine skin was 0.18 and 0.23 :g/cm2 , respectively (Fig. 1a). Increased MXD targeting to follicles was found after laser application. The increase of fluence did not significantly increase (p > 0.05) the follicular uptake of MXD. MXD accumulation in the follicles was shown to rise twofold upon laser treatment. DPC uptake in nude mouse follicles with 6- and 10-:m ablation resulted in a fourfold and sixfold enhancement compared with the control (Fig. 1b). Nevertheless, laser utilization did not increase DPCP deposition in porcine follicles (p > 0.05). The same result was observed for FITC–peptide uptake (Fig. 1c). The laser promoted FITC– peptide uptake in mouse follicles by twofold (6 :m) and sixfold (10 :m), whereas this enhancement was not revealed in porcine follicles. DOI 10.1002/jps.24143
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Figure 1. The follicular uptake (:g/cm2 ) of MXD (a), DPCP (b), and FITC–peptide (c) in nude mouse skin and porcine skin with and without Er–YAG laser treatment. *p < 0.05 compared with intact skin. All data represent the mean ± SD of four experiments.
Figure 2. Comparison of the skin deposition (:g/g) of MXD (a), DPCP (b), and FITC–peptide (c) in intact skin and sebum-removal skin after in vitro application on nude mouse skin and porcine skin with and without Er–YAG laser treatment (ablation depth of 6 :m). *p < 0.05 compared with intact skin. All data represent the mean ± SD of four experiments.
In Vitro Drug Accumulation in Sebum-Removal Skin Figure 2 illustrates the skin deposition of the permeants delivered via sebum-removal skin. The data above the column in Figure 2 indicate the ER of skin accumulation between sebumremoval skin and intact skin. Removal of sebum significantly increased (p < 0.05) MXD deposition in nontreated skin by a factor of approximately 4 (Fig. 2a). A lower ER was observed for the laser-treated group for both nude mouse and porcine skin. This phenomenon was also demonstrated for DPCP and FITC–peptide (Figs. 2b and 2c). The accumulation of DPCP and FITC–peptide in normal skin and sebum-removal skin was comparable (p > 0.05) after laser irradiation at a depth of 6 :m. Vertical Observation of Skin by Fluorescence Microscopy Fluorescence microscopy was employed to examine the distribution of dyes in skin with or without laser exposure. Rhodamine B and Nile red show log P values of 1.8 and 3.8, respectively. The log P of rhodamine B and Nile red simulates that of MXD (1.2) and DPCP (3.9). Both dyes could be the permeant substitutes of MXD and DPCP for exerting a strong fluorescence in the skin. Figure 3 shows the skin distribution of rhodamine B. The top panel is the H&E staining of porcine skin morphology. The fluorescence imaging of the vertical skin sections is observed in the bottom panel. The skin without any treatment is used as a negative control as shown in Figure 3a. The negative control displayed some autofluorescence from the epidermal layer. Nude mouse skin was not evaluated in this experiment because of its strong autofluorescence in the wavelengths used (data not shown). The fluorescence intensity in the epidermis seemed to be stronger than in the negative control after application of rhodamine B on intact skin (Fig. 3b). Laser treatment could strengthen the red fluorescence along the SC and epidermis (Fig. 3c). The fluorescent signal was mainly deDOI 10.1002/jps.24143
tected in intercellular regions of the epidermis (circle in Fig. 3c), suggesting a translocation of rhodamine B in tight junctions that are the paracellular regions of the epidermis. Tight junctions play an important capacity for epidermal barrier function. Some fluorescence diffused into the upper dermis (rectangle in Fig. 3c). Nile red fluorescence was observed by using the same filter set as with rhodamine B. Figures 4a and 4b show the Nile red fluorescence images obtained from the porcine skin of the control and the laser-exposed site, respectively. Fluorescence microscopy of intact skin revealed Nile red in the epidermis and hair follicles (Fig. 4a), indicating an important route of appendages for Nile red penetration. The laser-ablated skin appeared as a continuous and broad fluorescence band in the SC and epidermis, especially in the intercellular regions (Fig. 4b). There was a more widespread fluorescence in the dermis of laser-treated skin compared with that of intact skin (rectangle in Fig. 4b). This signal was able to approach the lower dermis. Figure 5 shows the images of porcine skin receiving FITC– peptide. No evidence of autofluorescence was found for the skin without any treatment by using an FITC filter set (Fig. 5a). FITC–peptide transport via intact skin was limited in the outermost SC with a very weak green signal (arrows in Fig. 5b). The fluorescence in laser-treated skin was distributed in SC and the epidermis in a continuous manner (Fig. 5c), whereas the dermis was not stained. The same as rhodamine B and Nile red, the observation of the epidermis exhibited the presence of FITC–peptide in intercellular sites. Horizontal Observation of Skin by CLSM In vivo skin accumulation of the dyes was examined by CLSM. This is an imaging technique to visualize skin samples at multiple depths parallel to the surface. A nude mouse was used as Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
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Figure 3. Fluorescence microscopy images of porcine skin with in vitro topical application of rhodamine B: (a) the skin without any treatment (blank), (b) the intact skin receiving rhodamine B, and (c) the laser-treated skin (ablation depth of 6 :m) receiving rhodamine B. The upper panel is the bright-field view of H&E-stained images; the lower panel is the fluorescence image.
Figure 4. Fluorescence microscopy images of porcine skin with in vitro topical application of Nile red: (a) the intact skin receiving Nile red and (b) the laser-treated skin (ablation depth of 6 :m) receiving Nile red. The upper panel is the bright-field view of H&E-stained images; the lower panel is the fluorescence image.
the model animal in this experiment because of the ease of handling. Figure 6 is a representative CLSM image of nude mouse skin receiving rhodamine, a fluorescence substitute of MXD. The top panel of this figure represents the collective signal of skin divided into 15 fragments. The bottom panel represents the fluorescence at a depth of 20 :m, which is the location of the nude mouse epidermis. No autofluorescence was observed for the blank skin at the wavelengths set for rhodamine B and Nile red (Fig. 6a). No significant deposition of red fluorescence was visualized for intact skin receiving rhodamine B (Fig. 6b). We Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
observed a stronger signal of rhodamine B in the full-thickness skin and the epidermal layer of the laser-treated group (Fig. 6c). The fluorescence distribution was homogeneous over the horizontal section of the skin. Nile red in intact skin showed a stronger fluorescence than rhodamine B (Fig. 7a), suggesting an easier partitioning of lipophilic permeant to the skin. As illustrated in Figure 7b, a more significant signal of Nile red was seen in the laserablated skin. The fluorescence could be mainly observed in the hair shafts (arrows in Fig. 7b) and the epidermis (the DOI 10.1002/jps.24143
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Figure 5. Fluorescence microscopy images of porcine skin with in vitro topical application of FITC–peptide: (a) the skin without any treatment (blank), (b) the intact skin receiving FITC–peptide, and (c) the laser-treated skin (ablation depth of 6 :m) receiving FITC–peptide. The upper panel is the bright-field view of H&E-stained images; the lower panel is the fluorescence image.
Figure 6. Confocal micrographs of nude mouse skin with in vivo topical administration of rhodamine B at an original magnification of 100x: (a) the skin without any treatment (blank), (b) the intact skin receiving rhodamine B, and (c) the laser-treated skin (ablation depth of 6 :m) receiving rhodamine B. The upper panel is a summary of 15 fragments at various skin depths; the lower panel is the fragment at the depth of 20 :m from skin surface.
bottom panel of Fig. 7b). CLSM set at the wavelengths for FITC showed a negligible autofluorescence of nude mouse skin (Fig. 8a). The greater signal from FITC–peptide was detectable in both full-thickness skin and the epidermis (Fig. 8b). An intense green fluorescence was detected in the skin irradiated by the Er–YAG laser (Fig. 8c). As shown in the bottom panel of Figure 8c, it seems that FITC–peptide is concentrated along the perifollicular region (arrows). A network of fluorescence could be visualized in the epidermis surrounding the follicular ducts. A good in vitro–in vivo correlation was DOI 10.1002/jps.24143
observed between the images of fluorescence microscopy and CLSM.
DISCUSSION Hair loss affects a large population of humans. Although the condition is never mortal, it causes deep social implications for the affected individual because of the significant change to the patient’s appearance. Many medications have been developed Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
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Figure 7. Confocal micrographs of nude mouse skin with in vivo topical administration of Nile red at an original magnification of 100x: (a) the intact skin receiving Nile red and (b) the laser-treated skin (ablation depth of 6 :m) receiving Nile red. The upper panel is a summary of 15 fragments at various skin depths; the lower panel is the fragment at the depth of 20 :m from skin surface.
for treating alopecia3,23 ; however, the therapeutic efficacy and successful rate are limited. A novel approach is needed to improve the therapies. We aimed to employ the Er–YAG laser to superficially ablate the skin to enhance the skin delivery of antialopecia actives. Porcine skin was used as the model permeation barrier in this study because of its similarity to human skin in terms of structure and thickness. In addition, nude mouse skin was selected as another skin model because of the features of alopecia skin. The scalp skin appears to show greater permeability than the other anatomical sites.24 Moreover, the lesional scalp of patients receiving alopecia leads to the loss of skin barrier function.25 It is well known that nude mouse skin has a higher permeability compared with intact human skin. The rodent skin may be feasible as a lesional skin model. Laser ablation increased skin accumulation of antialopecia drugs at different levels for both nude mouse and porcine skins. The same trend was observed for calculating flux values. Once a permeant has been diffused into the SC and superficial layer after laser treatment, it can subsequently be transported into a deeper skin strata and vasculature.26 The SC thickness of nude mouse skin is approximately 10 :m.27 A laser ablation depth of 6 and 10 :m could partly and completely strip the SC layers, respectively. Histological examination of porcine skin exhibits an SC thickness of 15–20 :m.28 Both laser fluences only ablated a limited amount of SC for porcine skin. Direct ablation and optical breakdown by a photomechanical wave (PW) are related to laser–skin interactions for promoting percutaneous absorption Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
of drugs. In ablation, laser irradiation elicits decomposition of the skin target into small fragments, which move away from the skin surface at a supersonic speed.29 This peeling can largely reduce the barrier function of the SC, thus facilitating drug passage into/through the skin. The PW is a broadband, unipolar, compressive wave generated by lasers. PW transiently permeabilizes the skin surface but cannot strip the SC. Lipid disruption in the SC is induced by PW to allow drug diffusion into deeper strata. Electron microscopy demonstrates an expansion of lacunar space within intercellular lipids of the SC and epidermis. On the basis of the previous investigation,30 this expansion produces transient pores that qualify drug delivery through the SC and into viable skin. The laser fluences used in this work reserved some SC layers. The PW may propagate into the remnant SC, increasing drug permeation. According to fluorescence microscopy imaging, the intercellular distribution of dyes in superficial skin after laser treatment indicates the partitioning or accumulation in the lipid bilayers. This confirms the PW interaction with SC lipids to disrupt the array of structures. Minoxidil is a hydrophilic permeant with a log P of 1.2. It is expected to show low-skin penetration because of the limited partitioning into the lipid bilayers. Exposure to laser treatment could enhance MXD permeation by controlled ablation and PW. The higher fluence generally resulted in greater delivery into/across the skin. The imaging also demonstrated that laser irradiation urged rhodamine B, the dye with a log P similar to MXD, to penetrate more deeply into the skin layers. DOI 10.1002/jps.24143
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Figure 8. Confocal micrographs of nude mouse skin with in vivo topical administration of FITC–peptide at an original magnification of 100x: (a) the skin without any treatment (blank), (b) the intact skin receiving FITC–peptide, and (c) the laser-treated skin (ablation depth of 6 :m) receiving FITC–peptide. The upper panel is a summary of 15 fragments at various skin depths; the lower panel is the fragment at the depth of 20 :m from skin surface.
MXD only shows pharmacological activity at the dermal papilla level.31 MXD cannot work to regrow hairs until it penetrates the skin deeply. A previous study32 reports that MXD is predominantly distributed in the outermost skin layers and only a small amount of MXD reaches viable skin. The use of laser treatment could conquer this limitation. Increasing MXD concentration in the skin would improve the response for hair growth and reduce the time required to see the results. Diphencyprone is a lipophilic agent with a log P of 3.9. Lipophilic permeant is anticipated to be diffused into lipid-rich SC with ease. SC ablation and disruption by the Er–YAG laser showed less enhancement on cutaneous delivery of DPCP than MXD. Another observation was that a deeper ablation depth (10 :m) did not further increase DPCP absorption compared with shallower ablation (6 :m). Loss of the lipid-rich SC by laser peeling may have retarded the partitioning of lipophilic permeant into the SC,33 decreasing the drug delivery into SC and offsetting the impact of permeation barrier disturbance by the laser. DPCP delivery should be deep enough to approach the immune cells near the follicles.34 The fluorescence observation of Nile red in laser-treated skin sections showed an abundant accumulation of this dye in the dermis and near the follicles. The problem of poor skin absorption of large hydrophilic peptides has led to difficulty in developing topical drug delivery systems. The experimental results demonstrated that FITC– peptide showed a negligible permeation across the skin. Laser exposure, especially the ablation depth of 10 :m, significantly enhanced percutaneous absorption of FITC–peptide. The flux with deeper ablation (10 :m) was about 10 times greater than that with shallower ablation (6 :m). The results of fluoresDOI 10.1002/jps.24143
cence microscopy and CLSM revealed that FITC–peptide was distributed relatively homogeneously in laser-treated skin over the nontreated skin. The facile interaction between the peptide and skin structure contributed to a great accumulation of peptide in laser-irradiated skin after its extensive passage into the skin.35 The tissues and cells around the hair follicles are regarded as the main site of action for antialopecia drugs. The results of follicular uptake suggest that the ablative laser raised drug delivery into the appendages. The superficial portion of the follicular infundibulum is lined by the epidermal layer with a well-developed SC and stratum granulosum.19 The laser could propagate the ablation and PW effects around the follicles. Accumulation of drugs in follicular tracts ensures a faster drug diffusion into a deeper skin strata.36 The evidence can be observed by fluorescence microscopy imaging of Nile red in the skin. Laser treatment increased the fluorescence distribution in both the follicles and the viable dermis. It is noticeable that laser irradiation enhanced DPCP and FITC–peptide uptake in the nude mouse follicles but not in the porcine follicles. According to previous studies,37,38 the follicles of nude/hairless mice show a degenerated status. The follicles are covered by dry sebum, cell debris, and desquamated corneocytes. The permeants have difficultly entering the inactive and closed follicles. Blume-Peytavi and Vogt39 suggest that topically applied drugs only penetrate active follicles. The laser may unfold the closed nude mouse follicles for efficient drug uptake. The percentage of closed follicles in porcine skin is minimal, resulting in the insignificant increase of permeant uptake. About 30%–50% of hair follicles in human skin are closed to penetration.36 Er–YAG Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
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laser treatment provided the opportunity to increase follicular targeting by exposing the inactive follicles. Appendageal pathways are especially preferred for hydrophilic permeants.40 On the contrary, Frum et al.41 demonstrate that permeants with log P of more than 2 showed inferior penetration into the follicles. Our results also showed that follicular MXD uptake in intact skin was greater than that of DPCP uptake. The targeting of DPCP to the follicles after laser treatment demonstrated a greater enhancement as compared with that of MXD, indicating that the laser was efficient in improving poor follicle delivery of a lipophilic substance. When considering dermal permeation of peptides, the appendageal pathway plays an essential role.42 A significant increase of FITC–peptide uptake by the follicles could be detected by laser ablation. One of the advantages for follicular accumulation is that this site offers a long-term reservoir for permeants in comparison to the SC.43 The laser may prolong therapeutic activity of antialopecia drugs. Sebum is composed of a lipid mixture synthesized in the pilosebaceous gland and excreted on the skin surface.44 Sebum on the skin surface can be characterized as the outermost part of the permeation barrier. It is important in playing a role of permeant partitioning from the vehicle to the SC. There is an abundant amount of sebum on scalp skin.24 Sebum removal produced a greater increment of MXD skin accumulation than DPCP and FITC–peptide. The sebum can act as a barrier to repel water and hydrophilic molecules.45 On the contrary, the presence of lipophilic sebum may favor the partitioning of lipophilic substances. The absence of sebum could remove the permeation barrier for hydrophilic MXD, thus raising skin deposition. The Er–YAG laser exhibited a higher enhancement on drug skin accumulation in the presence of sebum than the removal of sebum. This indicates that the laser interacted with sebum to improve drug penetration. As laser irradiation can interact with SC lipids to break up barrier function,16,46 this effect may also be observed for interaction with sebum, which has contents similar to SC lipids. Another possibility is that sebum removal can reduce skin hydration.24 Er:YAG laser irradiation with a wavelength of 2940 nm is highly absorbed by water. Insufficient water in the skin may diminish the ablation effect of the Er–YAG laser, thus reducing the drug permeation enhancement. Caution should be taken when considering the possible application of laser-assisted drug permeation because some laser modalities can affect hair growth. The 308-nm excimer laser and 655-nm LaserComb are proved to be efficient for treating androgenetic alopecia and patchy alopecia areata in the scalp.47–49 The 488-nm argon laser, 755-nm alexandrite laser, and 810-nm pulsed diode array are employed to remove hair.50 The Er–YAG laser emits a wavelength of 2940 nm, which targets water as a chromophore. It is expected that Er–YAG modality shows a minimal effect on hair growth because of its different wavelength compared with the lasers for growing or removing hairs. Another concern is the safety of the ablative laser for enhancing drug delivery. The laser fluences used for drug delivery are much less than those used for rejuvenation and scar removal. Even the SC of the mouse can be precisely ablated without damage to the underlying epidermis.51 As the SC rapidly regenerates, the skin can be recovered to a normal status within 1–3 days after treatment at low fluences as examined by transepidermal water loss and histology.16,17,28 Some SC layers were unaffected after a 6- or 10-:m laser ablaLee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
tion. This suggests the possibility of lateral migration of intact cells for fast re-epithelialization. The remaining unaffected tissues serve as a reservoir for healing.52 This leads to a quick wound repair process, which is like the concept of fractional resurfacing.53 Further study is needed to explore the possibility of this quick re-epithelialization by the remnant SC. Another benefit is that the SC remnants assure some integrity of skin defense for prohibiting infection and toxins.
CONCLUSIONS Alopecia is a scalp disease with a high prevalence nowadays. Because of the unsatisfactory outcomes of the current drug therapy, development of a new drug delivery strategy is necessary. In this study, we demonstrated that Er–YAG laser ablation was capable of promoting skin permeation of a series of antialopecia drugs. Laser treatment greatly facilitated skin absorption of MXD, DPCP, and FITC–peptide, so that it was detectable in the SC and epidermis. MXD permeation generally showed a greater enhancement by the laser as compared with DPCP and FITC–peptide. The experimental results also justify the ability of laser treatment to deliver the permeants into the follicles, the principal target of antialopecia drugs. The Er–YAG laser also propagated in the sebum, exerting the permeation enhancement ability. The results of the present work suggest a potential application of laser-assisted drug permeation for treating alopecia. These experimental profiles encourage us to further investigate the clinical efficacy of antialopecia drugs mediated by the Er–YAG laser.
ACKNOWLEDGMENT The authors are grateful for the financial support from Chang Gung University of Science and Technology (EZRPF3C0221).
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DOI 10.1002/jps.24143