Skin Research and Technology 2012; 18: 7076
Printed in Singapore
All rights reserved
doi: 10.1111/j.1600-0846.2011.00533.x
Novel anti-wrinkle effect of cosmeceutical product
with new retinyl retinoate microsphere using
biodegradable polymer
H. Kim1,2, M. Kim3, Y. Quan4, T. Moon5, J. Mun5, H. Cho2, N. Park2, W. Moon2, K. Lee2, H. Kim2,
J. Lee2, H. Ryoo2, and H. Jung1
1
Department of Biotechnology, Yonsei University, Shinchon-dong, Seoul, Republic of Korea, 2Department of Skin Science and Cosmetics, ENPRANI Co.,
Ltd., Shinhung-dong, Inchon, Republic of Korea, 3Department of Product Development, Samkyung Costech Co., Ltd., Shin-dong, Suwon-si, Kyunggi-do,
Republic of Korea, 4Department of Biopharmaceutics, Kyoto Pharmaceutical University, Nakauti-cho, Misasagi, Yamashina-ku, Kyoto, Japan, and
5
Department of Clinical Evaluation, Ellead Skin Evaluation center, Seohyun-dong, Seongnam-si, Kyunggi-do, Republic of Korea
Background: The novel hybrid retinoid, retinyl retinoate, is
a synthetic material that was designed to reduce the side
effects of retinoic acid and to increase the stability of retinol.
The formulation of the retinyl retinoate, however, is required
to enhance skin permeation, and thus to increase the anti-
wrinkle effect.
Aim: To identify the efficacy of retinyl retinoate microsphere
using biodegradable polymer as an anti-aging agent of
cosmetics in treating females over 30 years old with peri-
orbital wrinkles.
Methods: The retinyl retinoate microsphere was prepared
using the biodegradable polymer; polylactic acid (PLA). We
also conducted two clinical studies with a total of 44 Korean
women for 12 weeks. In the first clinical study, 20 patients
completed a 12-week trial of cream A [3% PLA-retinyl
retinoate (2%) microsphere] applied twice daily to the
face. In the second clinical study, 24 patients completed a
12-week trial of cream B (0.06% retinyl retinoate) applied
twice daily to the face. Efficacy was based on a global
photodamage score, photographs, and image analysis
using replicas and Visiometers every 4 weeks.
L IKE ALL organs, skin undergoes characteristic
changes with age. In addition, photoaging
due to UV radiation causes undesirable changes in
skin appearance (1). Photoaged skin is character-
ized by an increase of wrinkles, thickening, inelas-
ticity, dryness, roughness, shallowness, and
pigmentary mottling (2, 3). Although retinoids,
such as retinol and retinoic acid, have been con-
sidered a highly efficient anti-wrinkle agent
through the booming of high-performance cos-
metics, the side effects of retinoic acid and the
instability of retinol in light and oxygen were the
main hurdle for their application in the cosmetics
industry (47). Recently, a novel hybrid retinoid,
70
2011 John Wiley & Sons A/S
Skin Research and Technology
Results: The PLA-retinyl retinoate microsphere was more
effective for the permeation of retinyl retinoate than retinyl
retinoate in itself. The cream A, which contains 3% PLA-
retinyl retinoate (2%) microsphere, showed a statistically
significant improvement in facial wrinkles (Po0.05) in 20
volunteers after only 4 weeks of application in a clinical trial
test. The visual wrinkle improvement and the maximum
roughness improvement rate (R2) for cream A was 6.05%,
8.03% higher than that of cream B which contains 0.06%
retinyl retinoate, after 4 weeks.
Conclusion: Retinyl retinoate has a potent anti-wrinkle
activity, and the PLA-retinyl retinoate microsphere could
be a useful cosmeceutical product for anti-aging purposes.
Key words: anti-wrinkle biodegradable polymer retinoid
retinyl retinoate photoaging
& 2011 John Wiley & Sons A/S
Accepted for publication 16 February 2011
retinyl retinoate, was introduced to reduce the side
effects of retinoic acid and to increase the stability
of retinol by applying a condensation reaction
between retinol and retinoic acid (8). Although
this hybrid retinol derivative had enhanced ther-
mal stability and decreased photosensitivity, and
exhibited decreased cell toxicity compared with
that of retinal (8), innovative delivery systems with
real consumer-perceivable benefits required to en-
hance skin penetration as active ingredients.
Because the encapsulation technology offers
greater effectiveness, lower toxicity, and lasting
stability, it has attracted a great deal of attention
for the potential application in pharmaceutic and

cosmetic compounds (911). Although one of the
most common methods is by using liposome (12),
the liposomal formulation with retinoids is struc-
turally unstable in the presence of oxidation
products and results in leakage (13). The emul-
sion delivery systems can be used for retinoid,
however, the hydrophilic emulsion system (oil-
in-water) accelerates retinoid instability by the
oxidation in the outer water phase and liphophi-
lic emulsion system (water-in-oil) induces unde-
sirable properties such as oiliness, greasiness,
and stickiness when applied to the skin. To over-
come these problems for protection of substances
prone to oxidation or action by atmospheric
moisture (14), the porous polymeric system is
introduced as novel cosmetic delivery systems
(11). This system utilizes microentrapment tech-
nology, wherein the particles have an open,
porous structure, compared with the continuous
shell structure associated with microencapsula-
tion, which can control the release of active
ingredients, such as retinyl retinoate, onto the
epidermis (14, 15).
In this study, we developed retinyl retinoate
microsphere using the biodegradable polymer
polylactic acid (PLA) to enhance skin penetration
and skin wrinkle improvement on aged skin. The
morphology of these microsphere was analyzed
and their influence on permeation in vitro and
skin wrinkle improvement on aged skin was
evaluated.
Methods
Preparation of PLA-retinyl retinoate microsphere
The biodegradable polymer (PLA), cetostearyl
alcohol, and cholestearyl nonanoate were dis-
solved in dichloromethane as an organic solvent
(16). The retinyl retinoate was synthesized from
all-trans retinol and all-trans retinoic acid
through coupling esterification (8). The retinyl
retinoate was dissolved in canola oil and dis-
persed into the prepared polymer solution. After
emulsification with poly(vinylalcohol) in water,
the organic solvent was evaporated using a va-
cuum drying system (17, 18). The PLA-retinyl
retinoate microsphere was obtained through a
filtration, drying, and grinding process, and the
final concentration of retinyl retinoate was 2% in
the microsphere. A Malvern Mastersizer QAS3002
(Malvern Instruments Ltd. Worcestershire, UK)
wet system was used to analyze size distribution
of the PLA-retinyl retinoate microsphere and the
Novel anti-wrinkle effect of cosmeceutical product
0.5 g samples were dispersed in 50 mL ethanol.
The morphological characteristics of PLA-retinyl
retinoate microsphere were studied by using a
scanning electron microscope (SEM, Shimadzu,
Kyoto, Japan) with gold coating.
In vitro skin permeation
A Franz diffusion cell was used to analyze the
skin permeation of retinyl retinoate with human
cadaver skin (1921). Human cadaver skin was
purchased from IIAM (USA), and was treated
to a thickness of approximately 200 mm with
AESCULAP dermatome machine (GA643, Tut-
tlingen, Germany). Before the experiment, the
dermatome skin was cut to 30 mm of circle.
PLA-retinyl retinoate microsphere and retinyl
retinoate were applied on the stratum corneum
(S.C.) side with an administration amount of
10 mg/cm2 cream. Skin permeation experiments
were carried out with Franz diffusion cells hav-
ing a diffusion area of 3.14 cm2 and a volume of
2.4 mL, which were set into the permeation
equipment (TransView C12, Kyoto Japan). After
application of PLA-retinyl retinoate microsphere
and retinyl retinoate, human dermatome skin
pieces were then carefully mounted onto the Franz
diffusion cells with the S.C. side facing towards
donor side. The receptor compartments were filled
with the phosphate buffer system (PBS, pH 7.4)
and the temperature was maintained at 32 1C over
the 6 h permeation study (each test material used
four skin donors for the experiment).
Retinyl retinoate penetration in S.C.
After the permeation experiment, test materials
remaining on the surface of skin were carefully
removed by wiping with cotton. Then, a scotch
tape was used for stripping the S.C. by peeling 15
times. The first peeling tape was discarded be-
cause of the adhered material left on the surface
of S.C. 2nd14th times successive tape strips were
collected and put into 50 mL centrifuge tube.
Retinyl retinoate was extracted from these
stripped S.C. with 15 mL of ethanol with 1 h
sonication. The extraction solution was trans-
ferred to a 100 mL beaker. The solvent was
evaporated completely in 70 1C water bath, and
1 mL of mobile phase consisting of acetonitrile
and methanol (60:40, v/v) was used to re-dis-
solve the extracted retinyl retinoate. After centri-
fugation at 10,500 g for 5 min, retinyl retinoate in
the supernatant was determined with HPLC.
71
Kim et al.

Retinyl retinoate penetration in the epidermis guideline. In the first clinical study, 20 patients
and dermis (age range; 3749 years) completed a 12-week
The skin piece after tape stripping was cut into trial of cream A [3% PLA-retinyl retinoate (2%)
small pieces, and homogenized in 5 mL of dis- microsphere] applied twice daily to the face
tilled water. Fifteen milliliters of diethyl ether excluding two women who abandoned study
was added to the homogenated to extract retinyl procedures. In the second clinical study, 24 pa-
retinoate. After centrifugation at 2,600 g for 5 min, tients (age range: 3450 years) completed a 12-
the diethyl ether layer was collected, and trans- week trial of cream B (0.06% retinyl retinoate)
ferred to a 100 mL beaker. This operation was applied twice daily to the face.
repeated three times. Then, the solvent was
evaporated completely in 50 1C water bath, and
Treatment regimen
1 mL of mobile phase consisting of acetonitrile
The clinical study was a prospective, double
and methanol (60:40, v/v) was used to re-dis-
blind, randomized, and controlled. Independent
solve the extracted retinyl retinoate. After centri-
researchers listed 46 subjects in order of prece-
fugation at 10,500 g for 5 min, retinyl retinoate in
dence in a computer-generated randomization
the supernatant was determined with HPLC. In
list. For the selection of random permuted blocks,
addition, in order to confirm the substance from
a randomization code was developed using com-
skin itself which may influence the determination
puter random number generator. The block
of retinyl retinoate in HPLC chromatograph, the
lengths 4, 8, and 12 varied randomly. The subjects
skin without test material application was mea-
were divided into groups A and B with women
sured as blank by the same procedure mentioned
having an equal probability of assignment to the
above.
groups. Individuals in group A were tested with
cream A whereas individuals in group B were
tested with cream B. Clinical evaluations were
Patients
made at weeks 0 (baseline), 4, 8, and 12.
A total of 44 generally healthy Korean women
aged between 34 and 50 years were selected from
volunteers. All patients satisfied the inclusion Self-assessment questionnaire
criteria with periorbital wrinkles (Global photo- Subject self-assessments via questionnaire were
damage Score 26) (22), confirmed by a derma- made at weeks 0 (baseline), 4, 8, and 12. Subjects
tologists medical interview and physical scored changes as 0, no change; 1, mild; 2, good;
examination. In all, 20 patients had mild photo- or 3, excellent.
damage (grades 23 on a 07 scale), and 24
showed moderate photodamage (grades 46 on
Investigators assessment
a 07 scale). None of the women had used topical
Subjects periorbital wrinkles were evaluated
retinoids on treatment areas for at least 3 months
with a double-blind test by two dermatologists.
before this study. None of the women had under-
The dermatologists evaluated subjects periorbi-
gone wrinkle removal, or peeling procedures
tal wrinkles based on a global photodamage score
within 6 months before the study. None of the
(0, none; 1, none/mild; 2, mild; 3, mild/moder-
women were pregnant or breastfeeding, or had
ate; 4, moderate; 5, moderate/severe; 6, severe; 7,
atopic dermatitis, allergic diathesis, or hypersen-
very severe) at weeks 0 (baseline), 4, 8, and 12
sitive skin. All the subjects were informed by the
(22). If the dermatologists evaluations differed,
investigators about the study objectives, outlines,
low-grade efficacy and high-grade adverse effect
test methods, and the possible adverse effects.
were selected. Subjects periorbital wrinkles were
Subjects filled out their profile, Case Report
classified into eight grades. Adverse effects, such
Form, questionnaire, and signed the Informed
as erythema, edema, scaling, itching, stinging,
Consent Statement. The study was conducted in
burning, tightness, and prickling, were recorded
accordance with the Guideline for Functional
by the investigator.
Cosmetics [KFDA 11-1470000-000863-01; http://
www.kfda.go.kr/index3.html (last accessed 9
September 2009)], which require at least 20 pa- Image analysis using replicas and visiometers
tients for the statistical analysis. Forty-six patients Wrinkle improvement was evaluated by measur-
were divided into two studies satisfying the ing skin roughness and wrinkles using the

72
 Novel anti-wrinkle effect of cosmeceutical product

Skin-Visiometer SV 600 (Courage & Khazaka,

Koln, Germany) (23). Replicas of right
and left periorbital areas were taken at weeks 0
(baseline), 4, 8, and 12. Skin replicas of crows-feet
were obtained according to the technique re-
ported by Grove et al. (24). and analyzed with
Visiometer software. Light intensity was analyzed
with Lambert & Beers Law, and the degree of
skin wrinkle improvement was calculated (Iex 5 -
Iine kd), where Iex is the transmitted light inten-
sity, Iin is the unattenuated light intensity, k is the
Napierian absorption coefficient of the medium, d
is the thickness of the medium. Evaluations were
performed in the same location with the same
lighting at each visit. Parameters used in the
assessment of skin visiometer SV 600 were as
follows: R1, skin roughness; R2, maximum rough-
ness; R3, average roughness; R4, smoothness
depth; and R5, arithmetic average roughness.

Statistical data analysis

The changes from the baseline of wrinkle and
roughness parameters (R1, R2, R3, R4, and R5)
were evaluated. A statistically significant differ-
ence in efficacy was achieved (P value of o0.05)
by paired t-test. The statistical data analysis was
performed by the utilization of SPSS software
version 10.0 (SPSS Inc., Chicago, IL, USA).
Adverse effects
We assayed for erythema, edema, scaling, itching,
stinging, burning, tightness, and prickling.
Results
Microspheres morphology
SEM images of PLA and PLA-retinyl retinoate
microsphere are shown in Fig. 1 with a regular
spherical shape. The morphology of PLA-micro-
sphere surface depends on the retinyl retinoate
loading. Although PLA-microsphere revealed a
smooth outer surface, retinyl retinoate loading
showed smaller dent over the outer surface. The
size range of PLA-retinyl retinoate microsphere
was found to be D (n,0.1) 5 1.76 and D (n,0.9) 5
26.13 with the mean diameter of 12.87 mm.
Skin permeation effect
Figure 2 shows the penetration of retinyl retino-
ate in the S.C. and in the epidermis and dermis
after 6 h permeation study. Retinyl retinoate
cream and PLA-retinyl retinoate microsphere
Fig. 1. Scanning electron microscopic (SEM) images of polylactic acid
(PLA) microsphere and PLA-retinyl retinoate microsphere. The scale
bar is 10 mm. Gold coating; IC-50 (Shimadzu), SEM; Superscan SS-
550 (Shimadzu). The size range of PLA-retinyl retinoate microsphere
was found to be D (n,0.1) 5 1.76 mm and D (n,0.9) 5 26.13 mm with
the mean diameter of 12.87 mm.
cream showed 2.95% and 3.29% of retinyl retino-
ate were penetrated into the S.C., while 0.03%
and 0.23% of retinyl retinoate were penetrated
into the epidermis and dermis, respectively.
Although the penetration of retinyl retinoate in
the S.C. showed no significant difference with
retinyl retinoate itself, the penetration in deep
layers of skin (epidermis and dermis) with PLA-
retinyl retinoate microsphere was 7.7-fold higher
than with retinyl retinoate in itself.
Subject self-assessments
Subjects performed self-assessments of their own
skin with blinded evaluation. In the first clinical
study of cream A, 50% of subjects (out of 20)
reported slight improvement of wrinkles and
40% of subjects (out of 20) reported moderate or
significant improvement of wrinkles. Improve-
ment reports were not obtained from two subjects

73
Kim et al.
Fig. 2. The penetration of retinyl retinoate in the S.C. (a) and in the
epidermis and dermis (b) in human cadever skin.
Fig. 3. Changes in the visual grade in subjects treated with cream A
(3% PLA-retinyl retinoate (2%) sphere) and cream B (0.06% retinyl
retinoate) for 12 weeks.
(10%). In the second clinical study of cream B,
62.5% of subjects (out of 24) reported slight
improvement of wrinkles and 25% of subjects
(out of 24) reported moderate or significant im-
provement of wrinkles. Improvement reports
were not obtained from three subjects (12.5%).
Although subjects noted improvement with the
two creams after 12 weeks, the overall satisfac-
tion with cream A was higher than that of cream
B. Cream A and cream B were all safe products
74
that did not lead to allergic contact dermatitis or
irritant contact dermatitis.
Investigators assessment
In the first clinical study of cream A, the visual
wrinkle grade was significantly reduced from
3.50 to 3.25, 3.20, and 3.15 after 4, 8, and 12 weeks,
respectively (Po0.05). The visual wrinkle im-
provement rate of cream A was 7.14% (P 5 0.021),
8.57% (P 5 0.010), and 10.0% (P 5 0.0047) after 4,
8, and 12 weeks, respectively. In the second
clinical study of cream B, the visual wrinkle
grade was slightly reduced from 3.83 to 3.79,
3.71, and 3.42 after 4, 8, and 12 weeks, respec-
tively. The visual wrinkle improvement rate of
cream B was 1.09% (P 5 0.33), 3.26% (P 5 0.083),
and 10.87% (P 5 0.0005) after 4, 8, and 12 weeks,
respectively. Although cream A and B showed
10.0% and 10.87% visual wrinkle improvement
after 12 weeks, photodamage scores significantly
improved after only 4 weeks of application in
cream A. An example of the facial improvement
effect is shown in Figs 3 and 4.
Image analysis using replicas and visiometers
Replicas from the periorbital areas of group A and
B were taken at weeks 0 (baseline), 4, 8, and 12,
and were analyzed in terms of five parameters
using the visiometer. Replicas were analyzed with
the Visiometer software (Skin-Visiometer SV 600,
Courage & Khazaka, Germany). Figure 5 is a
comparison of improvement rate in the para-
meters at 4 and 12 weeks with cream A and cream
B. In cream A, statistically significant differences
in the values of R2 (maximum roughness) and R3
(average roughness) were observed (R2; 8.58%,
P 5 0.0099, R3; 6.55%, P 5 0.0024) at 4 weeks. At 12
weeks, statistically significant differences in the
values of R1 (skin roughness) and R4 (smoothness
depth) were also observed (R1; 12.32%, P 5 0.014,
R4; 18.94% P 5 0.0027). In cream B, statistically
significant differences in the values of R1 (skin
roughness), R2 (maximum roughness), R3 (aver-
age roughness) and R5 (arithmetic average rough-
ness) were also observed (R1; 9.41%, P 5 0.027, R2;
7.09% P 5 0.029, R3; 6.30% P 5 0.046 and R5;
13.52% P 5 0.031) after 12 weeks. This result com-
bined with the significant difference in the max-
imum roughness (R2) and average roughness (R3)
in cream A after 4 weeks, indicated faster and
greater effect of PLA-retityl retinoate microsphere
on wrinkle improvement.

Fig. 4. A patient with wrinkles before (a, c) and 12 weeks after (b, d) treatment with cream A (a, b) or cream B (c, d).
Fig. 5. Changes in wrinkle improvement were analyzed by a Skin
Visiometer SV600 after 4 weeks (a) and 12 weeks (b) with cream A or
cream B. Asterisk indicates statistical significance *Po0.05,
**Po0.01, and ***Po0.001. R1, skin roughness; R2, maximum
roughness; R3, average roughness; R4, smoothness depth; and R5,
arithmetic average roughness.
Novel anti-wrinkle effect of cosmeceutical product
Discussion
The retinyl retinoate of the biodegradable micro-
sphere (PLA-retinyl retinoate microsphere) was
more permeable than the retinyl retinoate in itself
in the Franz diffusion cell experiment. The percen-
tage penetration of retinyl retinoate in the deep
layers of skin (epidermis and dermis) with PLA
microspheres was 7.7-fold higher than with retinyl
retinoate in itself. Also, the topical application of
the retinyl retinoate cream with microspheres sig-
nificantly improved facial wrinkles (Po0.05) in 20
volunteers within 4 weeks. Ninety percent of sub-
jects reported slight or significant improvement of
wrinkles in self-assessment and statistically signifi-
cant difference in the value of R2 and R3 were
observed at only 4 weeks (25). The adverse effects,
such as erythema, edema, scaling, itching, stinging,
burning, tightness, and prickling were not reported
in this retinyl retinoate loaded PLA-microspheres.
Recently, a novel hybrid retinoid, retinyl re-
tinoate, was introduced to reduce the side effects
of retinoic acid and to increase the stability of
retinol and this hybrid retinol derivative had
enhanced thermal stability and decreased photo-
sensitivity (8). Innovative delivery systems, how-
ever, with real consumer-perceivable benefits
required to enhance skin penetration as active
ingredients. In this study, we showed the faster
wrinkle improvement using biodegradable poly-
mer microspheres. This encapsulation technology
75
Kim et al.

offers greater effectiveness and lasting stability
for retinyl retinoate which tend to decompose
expose to heat, light, and oxygen.
In summary, the formulation of microsphere
using a biodegradable polymer with retinyl re-
tinoate can enhance the permeability rate of the
retinoid component. Thus, PLA-microsphere
with retinyl retinoate can be conveniently used
as a useful cosmeceutical product for the preven-
tion and improvement of skin aging, and the
treatment of skin problems.
Acknowledgements
This work was supported by a grant from the
Korea Health & Medical Technology R&D Pro-
ject, Ministry of Health & Welfare (0405-CS00-
0501-0010) and Seoul R&BD Program (SS100001).

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Address:
H Jung
Department of Biotechnology
Yonsei University
Shinchon-dong
Seoul
Republic of Korea
Tel: 182 2 2123 2884
Fax: 182 2 362 7265
e-mail: hijung@yonsei.ac.kr

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