ORIGINAL ARTICLE

Fractional Carbon Dioxide Laser Treatment to Enhance

Skin Permeation of Ascorbic Acid 2-Glucoside with
Minimal Skin Disruption
CHIEN-YU HSIAO, MS,*†# CHUN-HSUN HUANG, PHD,‡# SINDY HU, MD, MS,§ YU-SHIEN KO, MD,
PHD,¶** HSIN-CHING SUNG, MS,†† CHIH-CHUN CHEN, MS,‡‡ AND SHIH-YI HUANG, PHD†

BACKGROUND Topical treatment with vitamin C has been used to treat photoaged skin and as a skin
whitener, but no standard procedure exists for percutaneous delivery.
OBJECTIVE To compare skin histology and the permeation of ascorbic acid 2-glucoside (AA2G) after
fractional and conventional carbon dioxide (CO2) laser pretreatment.
METHODS The effect on porcine skin of treatment with different strengths of fractional and conventional CO2
laser treatment was examined using scanning electron microscopy and transmission electron microscopy.
Permeation of AA2G through porcine skin was tested in vitro using a Franz diffusion chamber. In vivo changes
in fluorescein thiocyanate permeability in nude mice were examined using confocal laser scanning
microscopy.
RESULTS Fractional CO2 laser treatment with four or fewer passes caused less disruption than conventional
laser treatment at the same fluence. AA2G permeation using four passes of fractional laser treatment was
similar to that seen with conventional CO2 laser treatment of the same fluence. Changes in permeability and in
depth of permeation were higher with conventional than fractional laser treatment.
CONCLUSION Fractional CO2 laser treatment can cause similar transdermal delivery of AA2G to conven-
tional laser treatment with less skin disruption and a different pattern of histologic change.
The authors have indicated no significant interest with commercial supporters.

B ecause ascorbic acid (vitamin C) has antioxi-

dant action and increases collagen synthesis,
it has been used topically to treat photoaged skin.1,2
It has also been reported to decrease melanin
synthesis and has therefore been used topically as a
skin whitener.3–5 Ascorbic acid is a hydrophilic
compound and does not penetrate the stratum
corneum easily. Increasing skin permeability with
laser treatment has therefore been proposed as a
means of delivering vitamin C through the stratum
corneum to the epidermis and dermis, where its
actions take place.
Laser treatment has been shown to increase trans-
dermal delivery of a number of substances, including
peptides, photosensitizing agents, insulin, anticancer

*Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Taoyuan, Taiwan;
†
School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan; ‡Department of Cosmetic Science,
Chang Gung University of Science and Technology, Taoyuan, Taiwan; §Department of Dermatology, Aesthetic Medical
Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan; ¶Department of Medicine, First Cardiovascular Division,
Chang Gung Memorial Hospital, Taipei, Taiwan; **College of Medicine, Chang Gung University, Taoyuan, Taiwan;
††
Department of Anatomy, Chang Gung University, Taoyuan, Taiwan; ‡‡Microscopy Core Laboratory, Chang Gung
Memorial Hospital at Linko, Taoyuan, Taiwan
#
These two authors contributed equally to this work.

© 2012 by the American Society for Dermatologic Surgery, Inc.  Published by Wiley Periodicals, Inc. 
ISSN: 1076-0512  Dermatol Surg 2012;1–10  DOI: 10.1111/j.1524-4725.2012.02454.x

1
 EFFECT OF FRACTIONAL CO2 LASER
agents, and DNA,6–10 but recovery time after the
skin damage caused by this type of treatment can
take several days, even though the strength of the
laser pulses used for transdermal drug delivery is
lower than that used to treat photoaged skin. A
newer technique, fractional laser treatment, causes
microscopic holes in the skin in a grid pattern
instead of irradiating the entire skin surface and has
been used to treat acne scars and photoaged
skin.11,12 It might therefore be used to enhance the
permeation of ascorbic acid into the skin without the
degree of destruction caused by and the longer
recovery period needed with conventional laser
treatment.
There is no standard pretreatment conventional or
fractional laser procedure for ascorbic acid 2-
glycoside (AA2G) delivery. To lay the groundwork
for the development of a standard protocol for
topical delivery of AA2G, we compared ascending
strengths of fractional and conventional CO2 laser
treatment for skin damage, increased permeability,
and transdermal delivery of AA2G using skin from
pigs and nude mice.
Materials and Methods
Laser Assemblies
The fractional CO2 laser (150XJ; Sharplan Laser
Inc., Yokneam, Israel) has a wavelength of
10,600 nm. An articulated arm was used to deliver
the laser beam to the skin surface. The handpiece
was able to create microscopic columns of skin
ablation or microscopic treatment zones (MTZs).
Typical MTZs had a diameter of 150 lm. The
occupied area of one irradiation dot was approx-
imately 0.018 mm2. The dimension of the treat-
ment area of the handpiece was 12 by 12 mm. This
area had 169 irradiation dots (13 9 13). The
coverage of the MTZs was 2% of the treatment
area when one pulse of the laser was used
(0.018 mm29169/12 9 12 mm2). For this study,
the skin received from one to four passes at fluences
from 5 to 9 W. The operator rotated the handpiece
2 DERMATOLOGIC SURGERY
at different angles so that, when more than one pass
was used, the irradiated areas of the individual
pulses did not overlap. The change to conventional
laser treatment was achieved simply by changing
the handpiece of the laser. The spot size of the
conventional laser was 3 mm in diameter.
Animals
Female nude mice (Balb/c-nu strain; 8 weeks old)
were obtained from the National Laboratory Animal
Center (Taipei, Taiwan). The Animal Technology
Institute Taiwan (Miaoli, Taiwan) supplied patho-
gen-free pigs (1 week old). The Institutional Animal
Care and Use Committee of Chang Gung University
of Science and Technology reviewed and approved
the animal experiment protocol. This committee
confirmed that the animal experiment followed the
guidelines as set forth in the Guide for Laboratory
Animal Facilities and Care.
Animal Protocol
Porcine skin was used for scanning electron
microscopy (SEM), transmission electron micro-
scopy (TEM), and in vitro AA2G permeation stud-
ies. The pigs were euthanized, and ten 2- by 2-cm
portions of dorsal skin removed from each pig (one
for each laser condition) for laser treatment. TEM,
SEM, and diffusion assays were all performed on
skin from the same pig. Eight pigs were used in all.
For in vivo permeation experiments, nude mice were
treated with fractional or conventional lasers, and a
cylinder containing fluorescein isothiocyanate
(FITC) solution was attached to their back over the
irradiated area, as described in a subsequent section,
for a 2 hour incubation period. The mice were then
euthanized and the treated skin excised for confocal
laser scanning microscopy (CLSM).
Ideally, we would have used the same species for in
vivo and in vitro studies, but pigs were too expensive
and hard to handle for the in vivo studies, and mice
were too small to use for the in vitro studies, so we
had to compromise on this.

Laser Experimental Protocol
To discover the optimal balance between good
permeation and minimal destruction, we started
with the lowest wattage (5 W) and only one pass of
the fractional laser machine and compared the
results with those using a conventional laser at the
same wattage. We then increased the number of
passes with the fractional laser at that wattage until
four passes had been used. This protocol was then
repeated with the next wattage.
Ultrastructure Examination Using SEM
Excised porcine skin samples with and without
laser treatment were cut into appropriate-sized
cubes and immediately fixed at 4°C in 3% para-
formaldehyde and 2% glutaraldehyde in 0.1 M
cacodylate buffer (pH7.4) overnight, washed three
times with 0.1 M cacodylate and 7% sucrose
buffer for 15 minutes, postfixed with 2% osmium
tetroxide for 1 hour, washed three times as before,
and immersed in 0.5% aqueous uranyl acetate for
30 minutes. The specimens were then dehydrated in
graded concentrations of ethanol, transferred to
isoamyl acetate, and critical-point dried using
liquid CO2. The dried specimens were affixed with
gold–palladium in an ion coater and examined
using a scanning electron microscope (S-5000;
Hitachi, Tokyo, Japan).
Ultrastructure Examination Using TEM
Excised porcine skin samples with and without laser
treatment were cut into appropriate-sized cubes and
immediately fixed at 4°C in 2% paraformaldehyde
and 3% glutaraldehyde in 0.1 M cacodylate buffer
(pH 7.4) overnight, washed three times with 0.1 M
cacodylate and 7% sucrose buffer for 15 minutes,
postfixed with 2% osmium tetroxide for 24 hours,
and washed three times as before. The specimens
were then dehydrated in graded concentrations of
ethanol, embedded in an epon–epoxy mixture and
sectioned. Thin sections were double-stained with
uranyl acetate and lead citrate and examined using
TEM (H-7500; Hitachi, Tokyo, Japan).
HSIAO ET AL
In Vitro Permeation of AA2G
The diffusion cell used in this in vitro experiment
was a Franz diffusion assembly. A piece of excised
porcine dorsal skin was mounted in the apparatus
with the stratum corneum facing the donor com-
partment. After laser pretreatment, the skin surface
was wiped with a cotton swab several times. The
receptor compartment (5.5 mL) was filled with a pH
7.4 citrate-phosphate buffer. The donor compart-
ment (0.5 mL) contained AA2G (Sigma-Aldrich, St.
Louis, MO, USA) in a pH 7.4 citrate–phosphate
buffer. The available area of the side-by-side cell was
0.7854 cm2. The receptor compartment was main-
tained at 37°C, and the contents were stirred with a
magnetic bar at 600 rpm. At appropriate intervals,
300-lL aliquots of receptor medium were with-
drawn and immediately replaced with an equal
volume of fresh receptor solution. The length of the
sampling period was 12 hours. The amount of drug
in the receptor medium was determined using high-
performance liquid chromatography (HPLC).
HPLC Analytic Method
The samples were analyzed using an HPLC system
consisting of a Dionex UltiMate 3000 pump, a
Dionex UltiMate 3000 autosampler, and a Dionex
UltiMate 3000 ultraviolet detector (Dionex, Sunny-
vale, CA). A 15-cm-long, 4.6-mm inner diameter
Inertsil ODS-3V column (GL Science, Tokyo, Japan)
was used. For AA2G analysis, the mobile phase
consisted of 0.7 g of potassium dihydrogen phos-
phate dissolved in 1,000 mL of double-distilled
water adjusted to pH 3 with 0.1M citric acid. No
methanol was used. The mobile phase was used at a
flow rate of 1 mL/minute, and the ultraviolet
detector was set at a wavelength of 254 nm.
In Vivo Nude Mouse Skin Permeation of FITC
Examined Using CLSM
The dorsal skin of the nude mouse was first treated
using irradiation with a fractional or conventional
laser. Then the dorsal skin was glued (Instant Super
Glue, Kokuyo, Osaka, Japan) to a glass cylinder
2012 3
 EFFECT OF FRACTIONAL CO2 LASER

with an available area of 0.7854 cm2. FITC/pH 7.4

buffer (0.2 mL) at a concentration of 0.1% was
added to the cylinder. The application time of the
vehicle, 2 hours, was based on two previous stud-
ies.9,13 After euthanizing the mouse and excising the
skin on which vehicle was applied, the skin surface
was washed 10 times using a cotton cloth immersed
in methanol. The skin samples obtained were then
examined for FITC fluorescence using CLSM. The
skin thickness was optically scanned at approxi-
mately 8-lm increments through the Z-axis of a
Leica TCS SP2 confocal microscope (Manheim,
Germany). Optical excitation was performed using a
488-nm argon laser beam, and fluorescence emission
was detected at 590 nm.

Statistical Analysis

Data were graphed and analyzed using SigmaPlot

software (Systat Software, Inc., Chicago, IL).
Cumulative amount–time profiles of in vitro
transdermal permeation were graphed as a line
plot with mean and standard deviation (SD) for
each condition. Results of flux across porcine skin
were summarized as mean ± SD for each condi-
tion, and these results were compared using one-
way analysis of variance with a post hoc Duncan
test. Statistical assessments were considered signif-
icant at p < .05.

Results

Electron Microscopy

Figure 1 shows SEM of porcine skin with no

treatment (A), fractional laser treatment of four
passes at 5W (B), and conventional laser treatment
at 5 W (C). Untreated skin shows corneocytes
overlapping each other in an orderly manner. In
contrast, the stratum corneum has become irregular
and uneven after fractional or conventional laser
treatment.

TEM of untreated skin (Figure 2A) shows an intact, Figure 1. Scanning electron microscopy observations
(9300) of porcine skin (A) without any treatment, (B) with
even stratum corneum with little or no space fractional laser treatment at 5 W for four passes, and (C)
between the individual layers. The lower-dose conventional laser treatment at 5 W.

4 DERMATOLOGIC SURGERY
 HSIAO ET AL

Figure 2. Transmission electron microscopy observations of porcine skin (95,000) with (A) no laser treatment, (B) fractional
laser treatment at 5 W for four passes, (C) conventional laser treatment at 5 W, (D) fractional laser treatment at 9 W for four
passes, (E) conventional laser treatment at 9 W.

(4 passes, 5 W) fractional laser treatment shows
partial detachment and a wavy pattern of the upper
layers of the stratum corneum, with wide spaces
between these layers (Figure 2B). Conventional laser
treatment at this dose shows more-uniform and
extensive damage to the stratum corneum and a
lesser degree of widening between layers. Fractional
laser treatment with the 9-W dose results in removal
of more stratum corneum, bigger spaces between the
uppermost remaining layers than the 5-W dose, and
some destruction of the epidermis and dermis.
Conventional laser treatment with the 9-W dose
shows complete ablation of the stratum corneum
and general destruction and necrosis of the under-
lying tissue.
Cumulative Amount–Time Profiles for
Transdermal AA2G Flux
As AA2G diffused through the laser-treated skin of
the Franz diffusion apparatus, its concentration in
the receptor compartment rose steadily and in a
dose-dependent manner (Figure 3). No AA2G flux

2012 5
 EFFECT OF FRACTIONAL CO2 LASER

occurred across untreated skin. Flux across skin with

fractional laser treatment rose as the number of
passes increased. The flux with four passes of the
fractional laser equaled the flux of the conventional
laser for the two lower fluences (5 and 7 W) and was
slightly lower for the highest (9 W) fluence.

AA2G Fluxes and Enhancement Ratios

Table 1 shows the fluxes and enhancement ratios for

diffusion of AA2G through porcine skin treated with
fractional and conventional laser treatment of vary-
ing doses. For each fluence, the diffusion of AA2G
increased as the number of passes of the fractional
laser increased. When the number of passes reached
four, the flux enhancement ratios using the frac-
tional laser became similar to those seen with
conventional laser treatment of the same fluence.

In Vivo FITC Permeation Studies of Laser-

Treated Nude Mouse Skin

Figure 4 shows CLSM imaging of nude mouse skin

after fractional (4 passes) and conventional laser
treatment at fluences of 5 and 9 W. In untreated
skin, some FITC fluorescence is seen but only in the
upper layers. At four passes at 5 W, fractional laser-
treated skin shows fluorescence that is stronger and
located more deeply in the skin than that with no
treatment. Conventional laser treatment at this
fluence shows even more and deeper fluorescence
than fractional laser treatment. Fractional laser
treatment shows more fluorescence with 9-W than
5-W treatment, but only a small amount of fluores-
cence has reached the deeper skin layers. At 9 W, in
contrast, fluorescence after conventional laser treat-
ment has increased and moved almost entirely to
these deeper layers.

Discussion

When four passes of the fractional laser were used,
fractional CO2 laser treatment was equivalent to
conventional CO2 laser treatment. That is, it
achieved a similar AA2G flux and enhancement
ration as that seen with conventional CO2 laser
Figure 3. In vitro cumulative amount–time profiles of
topical delivery in porcine skin of ascorbic acid 2-glucoside
(AA2G) at fluences of (A) 5 W, (B) 7 W, (C) 9 W by fractional
laser treatment for one to four passes and conventional
treatment. Each value represents the mean ± standard
deviation (n = 8).

6 DERMATOLOGIC SURGERY
 HSIAO ET AL

TABLE 1. Ascorbic Acid 2-Glucoside Fluxes (Flux) and Enhancement Ratios (ERs) Across Porcine Skin After
Fractional and Conventional Treatment Using a Carbon Dioxide Laser (N = 8 for each condition)
Fractional Laser Treatment Conventional Laser Treatment

Flux, lg/cm2 per Flux, lg/cm2 per

Fluence (W) Passes, n hour, mean ± SD* ER† hour, mean ± SD* ER†
0 (no treatment) 0 0.94 ± 0.12a 1 0.94 ± 0.12a 1
5 1 34.07 ± 6.61b 36 83.53 ± 11.24c 89
2 47.73 ± 7.86b 51 NA
4 79.42 ± 10.14c 84 NA
7 1 57.55 ± 9.22b 61 95.18 ± 13.02cd 101
2 85.25 ± 10.57c 91 NA
4 93.16 ± 12.41cd 99 NA
9 1 82.35 ± 11.05c 88 127.93 ± 23.47d 136
2 116.99 ± 19.87d 124 NA
4 125.19 ± 22.56d 133 NA

*p < 0.05 through one-way analysis of variance test with a post-hoc Duncan test a<b<c<d.
†The flux of laser-pretreated group/flux of no-treatment group (fluence = 0 W and pass number = 0).
SD, standard deviation; NA, not assessed.

treatment with the same fluence, but the histologic
changes that the two treatments caused were not
equivalent. SEM of the skin surface was similar with
the two treatments (at 5 W), but TEM showed skin
with fractional laser treatment to have semidetached
layers of the stratum corneum with large spaces
between them, whereas conventionally treated skin
showed loss of most of the stratum corneum and
only small spaces between the remaining layers.
Permeation of the skin by FITC also showed a
different pattern, in that FITC permeated to deeper
skin layers after conventional laser treatment than
after fractional laser treatment.
seen with conventional laser treatment even when
six passes of the fractional laser were used.13 Skin is
not a homogeneous substance, having a barrier to
hydrophilic substances in the stratum corneum and
lipophilic and hydrophilic pathways below. In
addition, laser treatment destroys some of its struc-
ture and makes the remainder irregular, and it is
thought that, although removal of the stratum
corneum can increase transdermal flux by removing
the barrier to hydrophilic substances, its removal can
decrease transdermal flux in some cases, because it
may also serve as a reservoir for the substances being
transported.

AA2G flux showed normal kinetics; that is, it
increased in a linear manner with fractional and
conventional laser treatment as time and dose
increased. Other substances have not shown such
simple kinetics. For example, peptide delivery after
laser treatment was not linearly related to size,9 and
the flux of the anticancer drug 5-fluorouracil and the
photosensitizer 5-aminolevulinic acid across nude
mouse skin reached a maximum and then decreased
as the fluence of an erbium-doped yttrium aluminum
garnet laser increased (Er:YAG).7,10 Permeation of
5-aminoleulinic acid across nude mouse skin after
fractional laser treatment did not reach the levels
SEM, TEM, and CLSM are not quantitative meth-
ods, but in this study, they showed that fractional
laser treatment, in the doses used, did not cause the
degree of destruction that conventional treatment
caused and did not cause permeability changes in
skin tissue to the depth that conventional treatment
did, although this lesser destruction and penetration
did not result in a smaller flux of AA2G. Fractional
laser treatment also has a shorter recovery time than
conventional laser treatment.14 The implication of
these observations is that fractional laser treatment
has advantages over conventional laser treatment for
transdermal delivery of vitamin C.

2012 7
 EFFECT OF FRACTIONAL CO2 LASER

(A)

(B)

(C)

(D)

(E)

Figure 4. Confocal laser scanning microscopic (CLSM) micrographs of nude mouse skin after the in vivo topical
administration of fluorescein isothiocyanate through the skin by laser pretreatment: (A) no-treatment group, (B) fractional
laser treatment at 5 W for four passes, (C) conventional laser treatment at 5 W, (D) fractional laser treatment at 9 W for four
passes, (E) conventional laser treatment at 9 W (original magnification 9 20). The skin specimen was viewed using CLSM at
8-lm increments through the Z-axis. The images in the right side of the photographs of the 10 fragments are the sum of all
fragments.

Vitamin C is used in topical cosmetic formulations
as a skin whitener. Gels or other topical vitamin C
preparations have also been used to treat photoag-
ing, rhytides, and acne scars, the idea being that, if
vitamin C can penetrate to its site of action, it can
block the reactive oxygen species that are causing
the damage and increase collagen synthesis to make
the skin more normal in appearance.1,2 These trials
were long (3 and 6 months) and had the biggest
effect on fine wrinkles and rhytides, but it was not
clear how well vitamin C was penetrating into the
lower skin layers. In this regard, in our experiment,
no AA2G diffusion across normal, untreated skin
was seen, even after 2 hours of incubation.

8 DERMATOLOGIC SURGERY

Fractional laser treatment has the advantage of
increasing transdermal permeability with less
destruction of the stratum corneum and epidermis
than most other methods. The question is what
degree of permeability is needed for adequate
delivery of vitamin C in the clinical setting. A
modest degree of permeability might be enough for
reasonable therapeutic results.
Much more experimentation needs to be done to
determine the clinical situations in which fractional
laser treatment with ascorbic acid may be useful, but
it seems clear that this method of delivery will give
better ascorbic acid delivery to epidermal and
dermal skin layers than topical or oral treatment.
Little ascorbic acid penetrates below the stratum
corneum after topical treatment, and little orally
administered ascorbic acid reaches these skin layers,
because the capacity of the gastrointestinal active
transport system used for absorption limits oral
absorption, and the ascorbic acid that is absorbed is
quickly undergoes enzymatic and nonenzymatic
oxidation and is thus eliminated.15,16
The clinical usefulness of this greater penetration
and deeper absorption needs to be explored further.
Dermal absorption is not a great advantage when
ascorbic acid is used for skin whitening, unless the
dermis performed the function of a reservoir,
because most melanocytes, the target cell, are in the
epidermis, but the deeper penetration would provide
an advantage in clinical conditions, such as photo-
aging, where stimulation of collagen synthesis is
desired.
Another question to be answered is whether the
combination of fractional laser treatment and deeper
ascorbic acid delivery would cause unacceptable
toxicity. There is not much current information to
answer this question. Hwang and colleagues noted
that, although 4 months of daily topical application
of 25% vitamin C caused mild stinging, burning,
and erythema in some patients, no postinflammatory
hyperpigmentation was seen.17 A second study, in
which the concentration of topical vitamin C was
HSIAO ET AL
not specified, reported similar results,2 and a third
study, in which 5% vitamin C was used, mentioned
no side effects.1 Most studies of fractional laser
treatment use Er:YAG lasers, but one study of
fractional CO2 laser treatment stated that universal
postinflammatory hyperpigmentation is seen after
conventional CO2 laser treatment, although only
one of 10 subjects experienced postinflammatory
hyperpigmentation after fractional CO2 laser treat-
ment.18
Limitations of the study were that we were unable to
measure penetration depth for ascorbic acid in
fractional and conventional treatment. We also did
not perform a comparative analysis of the quanti-
tative uptake of ascorbic acid in the ex vivo pig and
in vivo nude mouse systems. We did not compare the
two systems in this way because we felt that a
quantitative comparison between two totally differ-
ent models would not be very meaningful, although
Hsaio and colleagues, in a recent study, examined
the flux of two ascorbic acid derivatives across nude
mouse skin given conventional CO2 treatment and
found this flux to be much greater.19
In conclusion, fractional CO2 laser treatment may
have potential clinical use for transdermal delivery
of vitamin C, but further investigation of this
possibility needs to be conducted.
Acknowledgments This work was supported by
Chang Gung Memorial Hospital Grants
(CMRPF190101).
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Address correspondence and reprint requests to: Shih-Yi
Huang, PhD, School of Nutrition and Health Sciences,
Taipei Medical University, 250 Wu-Hsing Street, Taipei
110, Taiwan, or e-mail: sihuang@tmu.edu.tw