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
2
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

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
of antialopecia drugs. C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3542–3552, 2014

Keywords: alopecia; minoxidil; diphencyprone; peptide; laser; absorption; percutaneous; skin; macromolecular drug delivery

INTRODUCTION include growth factors, proteins, genes, and fullerenes.7–9 Abun-

dant investigations document the use of peptides to counter
Alopecia is known as hair loss on the scalp because of an
the effect of alopecia. These include soymetide-4,10 formyl–
androgenetic process (androgenetic alopecia) or an inflamma-
methyonyl–leucyl–phenylalanine,11 prolactin,12 and calcitonin
tory process (alopecia areata). The prevalence of androgenetic
gene-related peptide.13 These biologics, however, show unsatis-
alopecia in Caucasian men is 96%.1 On the contrary, alope-
factory results in treating alopecia because of their instability
cia areata affects 2.1% of the population.2 Minoxidil (MXD) is
and molecular size, which is too large to penetrate the skin.
developed for the treatment of both alopecia types. This drug
Efficient topical drug delivery is challenging because of the
at a 5% dose is the first-line topical therapy for androgenetic
formidable barrier function of the stratum corneum (SC). SC re-
alopecia.3 It can reduce baldness by the mechanisms of va-
moval by tape-stripping, mechanical abrasion, and laser treat-
sodilation, enhanced proliferation, and angiogenesis. Diphen-
ment has shown to be effective for promoting drug permeation
cyprone (DPCP) is a contact allergen for topical immunother-
via the skin.14 The approaches of tape-stripping and abrasion
apy of alopecia areata. Alopecia treatment by drugs is always
are limited and lack reproducibility because of the poor abil-
inefficient. The effect of MXD is not permanent, and the treat-
ity to control the SC ablation level. Laser treatment can pre-
ment cessation contributes to hair loss in 4–6 months.4 More-
cisely and selectively remove SC in a controlled and noncon-
over, contact dermatitis occurs in 6% of patients receiving 5%
tact manner.15 The duration of laser irradiation is in the range
MXD.5 A lag time of 3 months from initiation of treatment
of nano- to microseconds, indicating a quick operation for en-
to initial hair growth is observed for DPCP with a success
hancing drug absorption.16 It has been shown that the ablative
rate of only 50%. The adverse effects of DPCP occur in 24%
lasers can increase topical delivery of small molecule drugs,
of patients.6 Recent advances in biotechnology have developed
macromolecules, and nanoparticles.17,18 The improvement of
macromolecules and nanoparticles for alopecia therapy. These
antialopecia drug delivery for achieving efficacious therapy is
urgent. We sought to evaluate the impact of ablative laser treat-
Correspondence to: Jia-You Fang (Telephone: +886-3-2118800; Fax: +886-3-
2118236; E-mail: fajy@mail.cgu.edu.tw) ment on cutaneous delivery of antialopecia drugs, including
Journal of Pharmaceutical Sciences, Vol. 103, 3542–3552 (2014) MXD, DPCP, and peptide. Hair follicles are the main target

C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association for these drugs in treating hair loss. The follicular openings of

3542 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 de-
livery 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 gly-
col (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 synthe-
sized 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). Spe-
cific pathogen-free pigs, 1-week-old, were provided by Animal
Technology Institute Taiwan (Miaoli, Taiwan). The animal ex-
perimental protocol was reviewed and approved by the Insti-
tutional Animal Care and Use Committee of Chang Gung Uni-
versity. 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 perme-
ants. The effective diffusion area between compartments was
DOI 10.1002/jps.24143
3543
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 spec-
trophotometry. The excitation and emission wavelengths were
set to 490 and 520 nm, respectively. The accumulation of per-
meants 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 flu-
oride (PVDF) membrane, the samples were detected by HPLC
or fluorescence spectrophotometry.
Hair Follicle Uptake
Differential stripping and cyanoacrylate skin surface cast-
ing 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 poly-
merized, 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 evap-
orate 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 ap-
plied 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 glyc-
erin 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 emis-
sion, 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 at-
tached 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 pe-
riod 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 wave-
lengths 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
3544 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 1. The In Vitro Skin Accumulation (:g/g) and Flux (:g/cm2 /h) into a deeper skin strata or circulation in an in vivo condi-
of MXD, DPCP, and Peptide via Nude Mouse Skin Treated with or tion. The cumulative amount of permeant in the receptor as a
Without Lasers function of time was calculated as flux, which followed in zero-
Laser Skin order fashion. As shown in Table 1, laser treatment delivered
Depth Accumulation Flux significantly (p < 0.05) more MXD into the skin than the non-
Drug (:m) (:g/g) ERSA (:g cm−2 h−1 ) ERflux treated group. The enhancement effect of the laser is shown in
Table 1 in terms of the enhancement ratio (ER). The laser
MXD 0 16.47 ± 3.88 – 1.45 ± 0.34 – demonstrated more than a twofold- and sixfold-greater skin ac-
6 40.14 ± 10.07 2.44 4.36 ± 1.10 3.01 cumulation of MXD via laser-treated skin at an ablation depth
10 106.82 ± 16.66 6.49 17.75 ± 2.24 12.24
of 6 and 10 :m than intact skin. A similar trend was revealed
DPCP 0 115.39 ± 20.06 – 7.35 ± 0.79 –
with flux values. Laser treatment followed by DPCP applica-
6 117.55 ± 35.28 1.02 7.21 ± 2.45 0.98
10 137.18 ± 26.84 1.19 9.29 ± 1.23 1.26 tion slightly increased skin accumulation and flux. However,
Peptide 0 1.15 ± 0.61 – 0 – this enhancement did not achieve a significant difference(p
6 1.59 ± 0.40 1.38 0.51 ± 0.08 – > 0.05) as compared with the control group. Laser ablation
10 2.24 ± 0.49 1.95 4.99 ± 0.98 – resulted in the enhancement of FITC–peptide accumulation
within the skin, although the ER was lower than that from
ERSA , enhancement ratio of the skin accumulation of laser-treated group/the
skin accumulation of untreated control group; ERflux , enhancement ratio of the MXD. The increment in ablation depth from 6 to 10 :m led to
flux of laser-treated group/the flux of untreated control group; –, not determined. a significant elevation in peptide skin deposition (ER: 1.4 vs.
Each value represents the mean and SD (n = 4). 2.0). The receptor amount of FITC–peptide by passive diffusion
was below the detection limit during a 24-h application. Laser
Table 2. The In Vitro Skin Accumulation (:g/g) and Flux (:g/cm2 /h) ablation of 6 and 10 :m to nude mouse skin resulted in an in-
of MXD, DPCP, and Peptide via Porcine Skin Treated with or Without
crease in FITC–peptide flux from 0 to 0.5 and 5.0 :g cm−2 h−1 ,
Lasers
respectively.
Laser Skin Table 2 shows drug permeation profiles of porcine skin. MXD
Depth Accumulation Flux deposition in the skin increased significantly (p < 0.05) from
Drug (:m) (:g/g) ERSA (:g cm−2 h−1 ) ERflux 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
MXD 0 1.03 ± 0.21 – 0.17 ± 0.05 –
6 9.03 ± 2.08 8.77 0.58 ± 0.10 3.41
(p > 0.05). MXD flux with laser treatment at 6 and 10 :m was
10 6.73 ± 1.53 6.53 0.89 ± 0.44 5.24 superior (p < 0.05) to that of the nontreated group with ER of
DPCP 0 11.38 ± 3.75 – 1.51 ± 0.36 – 3.4 and 5.2, respectively. In the porcine skin model, the laser
6 20.42 ± 3.75 1.79 5.90 ± 1.76 3.91 could improve the skin accumulation and the flux of DPCP (p
10 18.44 ± 3.51 1.62 5.72 ± 0.09 3.79 < 0.05). An increment of about 20-fold in DPCP skin deposition
Peptide 0 0.64 ± 0.23 – 0 – was found with laser treatment at both 6 and 10 :m. DPCP flux
6 0.68 ± 0.19 1.06 0.04 ± 0.01 – by laser resurfacing at 6 and 10 :m was fourfold higher than
10 1.07 ± 0.27 1.67 0.33 ± 0.10 – that of the nontreated control. With respect to FITC–peptide,
ERSA , enhancement ratio of the skin accumulation of laser-treated group/the an ablation depth of 6 :m was insufficient to enhance skin up-
skin accumulation of untreated control group; ERflux , enhancement ratio of the take (p > 0.05). Skin accumulation of FITC–peptide by laser
flux of laser-treated group/the flux of untreated control group; –, not determined. exposure at a 10-:m depth was 1.7 times greater than that of
Each value represents the mean and SD (n = 4).
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
emission were set to 488 and 510–540 nm for the skin receiving from 0.04 to 0.33 :g cm−2 h−1 .
FITC–peptide. Images were taken by summing 15 fragments
at different depths from the skin surface. Hair Follicle Uptake
Statistical Analysis It is important to increase the uptake of antialopecia drugs
Statistical analysis of differences between the groups was into the hair follicles. A differential stripping technique was
carried out using the Kruskal–Wallis test. The post-hoc test used to detect the drug amount selectively accumulated in the
used for checking individual differences was Dunn’s test. A follicles as depicted in Figure 1. The recovery of MXD from
0.05 level of probability (p < 0.05) was taken as the level of casts of intact nude mouse skin and porcine skin was 0.18 and
significance. 0.23 :g/cm2 , respectively (Fig. 1a). Increased MXD targeting to
follicles was found after laser application. The increase of flu-
ence did not significantly increase (p > 0.05) the follicular up-
RESULTS take of MXD. MXD accumulation in the follicles was shown to
rise twofold upon laser treatment. DPC uptake in nude mouse
In Vitro Skin Permeation
follicles with 6- and 10-:m ablation resulted in a fourfold and
An in vitro percutaneous absorption was first performed to sixfold enhancement compared with the control (Fig. 1b). Nev-
evaluate the effect of laser irradiation on skin transport of ertheless, laser utilization did not increase DPCP deposition
antialopecia drugs. Delivery of drugs into/across nude mouse in porcine follicles (p > 0.05). The same result was observed
skin and porcine skin is summarized in Tables 1 and 2. The for FITC–peptide uptake (Fig. 1c). The laser promoted FITC–
drug retained in the skin reservoir dictates accumulation to peptide uptake in mouse follicles by twofold (6 :m) and sixfold
the superficial skin layer, whereas the permeant received in (10 :m), whereas this enhancement was not revealed in porcine
the receptor compartment simulates the extent of diffusion follicles.

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014 DOI 10.1002/jps.24143

 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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 de-
livered via sebum-removal skin. The data above the column in
Figure 2 indicate the ER of skin accumulation between sebum-
removal 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 distri-
bution of dyes in skin with or without laser exposure. Rho-
damine B and Nile red show log P values of 1.8 and 3.8, re-
spectively. The log P of rhodamine B and Nile red simulates
that of MXD (1.2) and DPCP (3.9). Both dyes could be the per-
meant 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 wave-
lengths 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 de-
DOI 10.1002/jps.24143
3545
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 junc-
tions 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 out-
ermost 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 mul-
tiple depths parallel to the surface. A nude mouse was used as
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
3546 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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 han-
dling. 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
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 hori-
zontal 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 laser-
ablated skin. The fluorescence could be mainly observed in
the hair shafts (arrows in Fig. 7b) and the epidermis (the

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014 DOI 10.1002/jps.24143

 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 3547

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 irradi-
ated by the Er–YAG laser (Fig. 8c). As shown in the bottom
panel of Figure 8c, it seems that FITC–peptide is concen-
trated 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
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

DOI 10.1002/jps.24143 Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014

3548 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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 im-
prove 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 More-
over, 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, re-
spectively. 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 opti-
cal breakdown by a photomechanical wave (PW) are related to
laser–skin interactions for promoting percutaneous absorption
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 pas-
sage into/through the skin. The PW is a broadband, unipolar,
compressive wave generated by lasers. PW transiently perme-
abilizes the skin surface but cannot strip the SC. Lipid dis-
ruption in the SC is induced by PW to allow drug diffusion
into deeper strata. Electron microscopy demonstrates an ex-
pansion 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 distribu-
tion of dyes in superficial skin after laser treatment indicates
the partitioning or accumulation in the lipid bilayers. This con-
firms 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 treat-
ment could enhance MXD permeation by controlled ablation
and PW. The higher fluence generally resulted in greater deliv-
ery into/across the skin. The imaging also demonstrated that
laser irradiation urged rhodamine B, the dye with a log P sim-
ilar to MXD, to penetrate more deeply into the skin layers.

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014 DOI 10.1002/jps.24143

 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 3549

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 predomi-
nantly 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 pep-
tides 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 fluores-
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 pep-
tide 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 fol-
licular 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. Ac-
cumulation of drugs in follicular tracts ensures a faster drug
diffusion into a deeper skin strata.36 The evidence can be ob-
served 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. Accord-
ing 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 perme-
ants 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

DOI 10.1002/jps.24143 Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014

3550 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
laser treatment provided the opportunity to increase follicular
targeting by exposing the inactive follicles.
Appendageal pathways are especially preferred for hy-
drophilic permeants.40 On the contrary, Frum et al.41 demon-
strate that permeants with log P of more than 2 showed in-
ferior 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 ef-
ficient in improving poor follicle delivery of a lipophilic sub-
stance. 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 de-
tected 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 Se-
bum 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 re-
moval 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 de-
position. 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 ir-
radiation 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 ap-
plication 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 treat-
ing 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 nor-
mal 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 abla-
Lee 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 tis-
sues 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 nec-
essary. In this study, we demonstrated that Er–YAG laser ab-
lation 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 gener-
ally 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 per-
meation enhancement ability. The results of the present work
suggest a potential application of laser-assisted drug perme-
ation for treating alopecia. These experimental profiles encour-
age 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).
REFERENCES
1. Chin EY. 2013. Androgenetic alopecia (male pattern hair loss) in
the United States: What treatments should primary care providers
recommend? J Am Assoc Nurse Pract 25:395–401.
2. Mirzoyev SA, Schrum AG, Davis MDP, Torgerson RR. 2014. Life-
time incidence risk of alopecia areata estimated at 2.1% by Rochester
Epidemiology Project, 1990–2009. J Invest Dermatol 134:1141–1142.
3. Tsuboi R, Itami S, Inui S, Ueki R, Katsuoka K, Kurata S, Kono T,
Saito N, Manabe M, Yamazaki M. 2011. Guidelines for the management
of androgenetic alopecia (2010). J Dermatol 38:1–8.
4. Mura S, Manconi M, Sinico C, Valenti D, Fadda AM. 2009. Pene-
tration enhancer-containing vesicles (PEVs) as carriers for cutaneous
delivery of minoxidil. Int J Pharm 380:72–79.
5. Alkhalifah A, Alsantali A, Wang E, McElwee KJ, Shapiro J. 2010.
Alopecia areata update. Part II. Treatment. J Am Acad Dermatol
62:191–202.
6. El-Zawahry BM, Bassiouny DA, Khella A, Zaki NS. 2010. Five-year
experience in the treatment of alopecia areata with DPC. J Eur Acad
Dermatol Venereol 24:264–269.
7. Zhou Z, Lenk R, Dellinger A, MacFarland D, Kumar K, Wilson
SR, Kepley CL. 2009. Fullerene nanomaterials potentiate hair growth.
Nanomed Nanotechnol Biol Med 5:202–207.
8. Takikawa M, Nakamura S, Nakamura S, Ishirara M, Kishimoto S,
Sasaki K, Yanagibayashi S, Azuma R, Yamamoto N, Kiyosawa T. 2011.
Enhanced effect of platelet-rich plasma containing a new carrier on
hair growth. Dermatol Surg 37:1721–1729.
DOI 10.1002/jps.24143
 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
9. Paik SH, Yoon JS, Ryu HH, Lee JY, Shin CY, Min KH, Jo SJ,
Kim KH, Kwon O. 2013. Pretreatment of epidermal growth factor pro-
motes primary hair recovery via the dystrophic anagen pathway after
chemotherapy-induced alopecia. Exp Dermatol 22:496–499.
10. Tsuruki T, Takahata K, Yoshikawa M. 2005. Anti-alopecia mecha-
nisms of soymetide-4, an immunostimulating peptide derived from soy
$-conglycinin. Peptides 26:707–711.
11. Tsuruki T, Yoshikawa M. 2006. Orally administered FPRL1 re-
ceptor agonist peptide MMK-1 inhibits etoposide-induced alopecia by
a mechanism different from intraperitoneally administered MMK-1.
Peptides 27:820–825.
12. Foitzik K, Langan EA, Paus R. 2009. Prolactin and the skin: A
dermatological perspective on an ancient pleiotropic peptide hormone.
J Invest Dermatol 129:1071–1087.
13. Kinori M, Bertolini M, Funk W, Samuelov L, Meyer KC, Emelianov
VU, Hasse S, Paus R. 2012. Calcitonin gene-related peptide (CGRP)
may award relative protection from interferon-(-induced collapse of
human hair follicle immune privilege. Exp Dermatol 21:221–226.
14. Lee WR, Shen SC, Liu CJ, Fang CL, Hu CH, Fang JY. 2006. Er-
bium:YAG laser-mediated oligonucleotide and DNA delivery via the
skin: An animal study. J Control Release 115:344–353.
15. Tsai TH, Jee SH, Chan JY, Lee JN, Lee WR, Dong CY. 2009. Vi-
sualizing laser-skin interaction in vivo by multiphoton microscopy. J
Biomed Opt 14:024034.
16. Gómez C, Costela Á, Garcı́a-Moreno I, Llanes F, Teijón JM, Blanco
D. 2008. Laser treatments on skin enhancing and controlling transder-
mal delivery of 5-fluorouracil. Lasers Surg Med 40:6–12.
17. Lee WR, Pan TL, Wang PW, Zhuo RZ, Huang CM, Fang JY. 2008.
Erbium:YAG laser enhances transdermal peptide delivery and skin
vaccination. J Control Release 128:200–208.
18. Genina EA, Bashkatov AN, Dolotov LE, Maslyakova GN, Kochubey
VI, Yaroslavsky IV, Altshuler GB, Tuchin VV. 2013. Transcutaneous
delivery of micro- and nanoparticles with laser microporation. J Biomed
Opt 18:111406.
19. Wosicka H, Cal K. 2010. Targeting to the hair follicles: Current
status and potential. J Dermatol Sci 57:83–89.
20. Campbell CSJ, Contreras-Rojas LR, Delgado-Charro MB, Guy RH.
2012. Objective assessment of nanoparticle disposition in mammalian
skin after topical exposure. J Control Release 162:201–207.
21. Aljuffali IA, Sung CT, Shen FM, Huang CT, Fang JY. 2014. Squar-
ticles as a lipid nanocarrier for delivering diphencyprone and minox-
idil to hair follicles and human dermal papilla cells. AAPS J 16:140–
150.
22. Teichmann A, Jacobi U, Ossadnik M, Richter H, Koch S, Sterry W,
Lademann J. 2005. Differential stripping: Determination of the amount
of topically applied substances penetrated into the hair follicles. J In-
vest Dermatol 125:264–269.
23. Gilhar A, Etzioni A, Paus R. 2012. Alopecia areata. N Engl J Med
366:1515–1525.
24. O’goshi KI, Iguchi M, Tagami H. 2000. Functional analysis of the
stratum corneum of scalp skin: Studies in patients with alopecia areata
and androgenetic alopecia. Arch Dermatol Res 292:605–611.
25. Betz RC, Pforr J, Flaquer A, Redler S, Hanneken S, Eigelshoven S,
Kortüm AK, Tüting T, Lambert J, De Weert J, Hillmer AM, Schmael
C, Wienker TF, Kruse R, Lutz G, Blaumeiser B, Nöthen MM. 2007.
Loss-of-function mutations in the filaggrin gene and alopecia areata:
Strong risk factor for a severe course of disease in patients comorbid
for atopic disease. J Invest Dermatol 127:2539–2543.
26. Lee S, McAuliffe DJ, Kollias N, Flotte TJ, Doukas AG. 2001. Perme-
abilization and recovery of the stratum corneum in vivo: The synergy
of photomechanical waves and sodium lauryl sulfate. Lasers Surg Med
29:145–150.
27. Lee WR, Shen SC, Pai MH, Yang HH, Yuan CY, Fang JY. 2010. Frac-
tional laser as a tool to enhance the skin permeation of 5-aminolevulinic
acid with minimal skin disruption: A comparison with conventional er-
bium:YAG laser. J Control Release 145:124–133.
28. Lee WR, Shen SC, Al-Suwayeh SA, Yang HH, Yuan CY, Fang
JY. 2011. Laser-assisted topical drug delivery by using a low-fluence
DOI 10.1002/jps.24143
3551
fractional laser: Imiquimod and macromolecules. J Control Release
153:240–248.
29. Yu J, Kalaria DR, Kalia YN. 2011. Erbium:YAG fractional laser
ablation for the percutaneous delivery of intact functional therapeutic
antibodies. J Control Release 156:53–59.
30. Doukas AG, Kollias N. 2004. Transdermal drug delivery with a
pressure wave. Adv Drug Deliv Rev 56:559–579.
31. Rossi A, Cantisani C, Melis L, Iorio A, Scali E, Calvieri S. 2012.
Minoxidil use in dermatology, side effects and recent patents. Recent
Pat Inflamm Allergy Drug Discov 6:130–136.
32. Padois K, Cantiéni C, Bertholle V, Bardel C, Pirot F, Falson F.
2011. Solid lipid nanoparticles suspension versus commercial solutions
for dermal delivery of minoxidil. Int J Pharm 416:300–304.
33. Chen WY, Fang CL, Al-Suwayeh SA, Yang HH, Li YC, Fang JY.
2013. Risk assessment of excess drug and sunscreen absorption via skin
with ablative fractional laser resurfacing: Optimization of the applied
dose for postoperative care. Lasers Med Sci 28:1363–1374.
34. Lin YK, Al-Suwayeh SA, Leu YL, Shen FM, Fang JY. 2013.
Squalene-containing nanostructured lipid carriers promote percuta-
neous absorption and hair follicle targeting of diphencyprone for treat-
ing alopecia areata. Pharm Res 30:435–446.
35. Abla N, Naik A, Guy RH, Kalia YN. 2005. Effect of charge and
molecular weight on transdermal peptide delivery by iontophoresis.
Pharm Res 22:2069–2078.
36. Rancan F, Papakostas D, Hadam S, Hackbarth S, Delair T, Primard
C, Verrier B, Sterry W, Blume-Peytavi U, Vogt A. 2009. Investigation
of polylactic acid (PLA) nanoparticles as drug delivery systems for local
dermatotherapy. Pharm Res 26:2027–2036.
37. Fang JY, Lee WR, Shen SC, Wang HY, Fang CL, Hu CH. 2004.
Transdermal delivery of macromolecules by erbium:YAG laser. J Con-
trol Release 100:75–85.
38. Shim J, Kang HS, Park WS, Han SH, Kim J, Chang IS. 2004.
Transdermal delivery of minoxidil with block copolymer nanoparticles.
J Control Release 97:477–484.
39. Blume-Peytavi U, Vogt A. 2011. Human hair follicle: Reservoir func-
tion and selective targeting. Br J Dermatol 165 (Suppl 2):13–17.
40. Mitragotri S. 2003. Modeling skin permeability to hydrophilic and
hydrophobic solutes based on four permeation pathways. J Control
Release 86:69–92.
41. Frum Y, Bonner MC, Eccleston GM, Meidan VM. 2007. The in-
fluence of drug partition coefficient on follicular penetration: In vitro
human skin studies. Eur J Pharm Sci 30:280–287.
42. Sachdeva V, Zhou Y, Banga AK. 2013. In vivo transdermal deliv-
ery of leuprolide using microneedles and iontophoresis. Curr Pharm
Biotechnol 14:180–193.
43. Lademann J, Richter H, Schaefer UF, Blume-Peytavi U, Teichmann
A, Otberg N, Sterry W. 2006. Hair follicles a long-term reservoir for
drug delivery. Skin Pharmacol Physiol 19:232–236.
44. Camera E, Ludovici M, Galante M, Sinagra JL, Picardo M. 2010.
Comprehensive analysis of the major lipid classes in sebum by rapid
resolution high-performance liquid chromatography and electrospray
mass spectrometry. J Lipid Res 51:3377–3388.
45. Tsai JC, Lu CC, Lin MK, Guo JW, Sheu HM. 2012. Effects of sebum
on drug transport across the human stratum corneum in vivo. Skin
Pharmacol Physiol 25:124–132.
46. Liu C, Zhang J, Yue Y, Luo Q, Zhu D. 2013. 1064-nm-Nd:YAG lasers
with different output modes enhancing transdermal delivery: Physical
and physiological mechanisms. J Biomed Opt 18:061228.
47. Al-Mutairi N. 2007. 308-nm excimer laser for the treatment of
alopecia areata. Dermatol Surg 33:1483–1487.
48. Leavitt M, Charles G, Heyman E, Michaels D. 2009. HairMax
LaserComb laser phototherapy device in the treatment of male andro-
genetic alopecia: A randomized, double-blind, sham device-controlled,
multicentre trial. Clin Drug Invest 29:283–292.
49. Wikramanayake TC, Rodriguez R, Choudhary S, Mauro LM, Nouri
K, Schachner LA, Jimenez JJ. 2012. Effects of the Lexington Laser-
Comb on hair regrowth in the C3H/HeJ mouse model of alopecia areata.
Lasers Med Sci 27:431–436.
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014
3552 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

50. Ibrahimi OA, Avram MM, Hanke CW, Kilmer SL, Anderson RR.
2011. Laser hair removal. Dermatol Ther 24:94–107.
51. Weiss R, Hessenberger M, Kitzmüller S, Bach D, Weinberger EE,
Krautgartner WD, Hauser-Kronberger C, Malissen B, Boehler C, Kalia
YN, Thalhamer J, Scheiblhofer S. 2012. Transcutaneous vaccination
via laser microporation. J Control Release 162:391–399.
52. Rokhsar CK, Fitzpatrick RE. 2005. The treatment of melasma with
fractional photothermolysis: A pilot study. Dermatol Surg 31:1645–
1650.
53. Goldberg DJ, Berlin AL, Phelps R. 2008. Histologic and ultrastruc-
tural analysis of melasma after fractional resurfacing. Lasers Surg Med
40:134–138.

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3542–3552, 2014 DOI 10.1002/jps.24143