Académique Documents
Professionnel Documents
Culture Documents
Gabriel Serrano 1,* Abstract: Liposomes have been intensively investigated as carriers for different applications
Patricia Almudéver 2,* in dermatology and cosmetics. Ascorbic acid has potent antioxidant and anti-inflammatory
Juan-Manuel Serrano 1 properties preventing photodamage of keratinocytes; however, due to its instability and low
Javier Milara 2–5 skin penetration, an appropriate carrier is mandatory to obtain desirable efficacy. The present
For personal use only.
Ana Torrens 1 work investigates the ability of a specific ascorbate phosphatidylcholine (PC) liposome to
overcome the barrier of the stratum corneum and deliver the active agent into the dermis to
Inmaculada Expósito 1
prevent photodamage. Abdominal skin from ten patients was used. Penetration of PC liposomes
Julio Cortijo 2–5
was tested ex vivo in whole skin, epidermis, and dermis by means of fluorescein and sodium
1
Sesderma Laboratorios, 2Department ascorbate. Histology and Franz diffusion cells were used to monitor the percutaneous absorption.
of Pharmacology, Faculty of Medicine,
University of Valencia, 3Clinical Ultraviolet (UV)-high performance liquid chromatography was used to analyze diffusion of
Research Unit, University General sodium ascorbate through the different skin layers, while spectrofluorimetry and fluorescent
Hospital Consortium, 4CIBERES,
microscopy were used for fluorescein monitoring. UVA/UVB irradiation of whole skin was
Health Institute Carlos III, 5Research
Foundation of the University General applied to analyze the antioxidant capacity by Trolox assay and anti-inflammatory effects by
Hospital of Valencia, Valencia, Spain tumor necrosis factor alpha and interleukin 1 beta enzyme-linked immunoassay. PC liposomal
*These authors contributed equally to formulation improved skin penetration of fluorescein and ascorbate. Fluorescein PC liposomes
this work showed better diffusion through epidermis than dermis while ascorbate liposomes showed better
diffusion through the dermis than the epidermis. Ascorbate PC liposomes showed preventive
antioxidant and anti-inflammatory properties on whole human skin irradiated with UVA/UVB.
In summary, ascorbate PC liposomes penetrate through the epidermis and allow nonstable
hydrophilic active ingredients reach epidermis and dermis preventing skin photodamage.
Keywords: skin absorption, liposomes, phosphatidylcholine, sodium ascorbate, fluorescein
Introduction
Skin is an organ subjected to a high degree of oxidative stress from both endogenous
(metabolism, inflammation) and exogenous sources (radiation, smoking, chemical
air pollutants, wind, etc), the most important being ultraviolet (UV) radiation. The
induction of oxidative damage by UV radiation has been demonstrated to occur in
lipids, proteins, and DNA.1 Adverse reactions include sunburn in short-term exposure,
Langerhans cell depletion, and local immunosuppression caused by longer UV exposure
and long-term effects such as cutaneous photoaging and skin cancer. As free radicals
Correspondence: Javier Milara can inflict cellular damage, organisms have evolved several lines of defense to protect
Clinical Research Unit, University General
Hospital Consortium, Avenida tres
cells from free radicals and repair DNA damage.2 In skin, the most important lines of
cruces number 2, 46014, Valencia, Spain defense are enzymatic (eg, superoxide dismutase, catalase, peroxidase) and nonenzy-
Tel +34 6 2023 1549
Fax +34 9 6197 2145
matic (eg, glutathione, α-tocopherol or vitamin E, ascorbate or vitamin C, β-carotene
Email xmilara@hotmail.com and ubiquinone) antioxidant systems. As the capacity of these systems is limited and
submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dermatology 2015:8 591–599 591
Dovepress © 2015 Serrano et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0)
http://dx.doi.org/10.2147/CCID.S90781
License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on
how to request permission may be found at: http://www.dovepress.com/permissions.php
Powered by TCPDF (www.tcpdf.org)
Serrano et al Dovepress
they can be overwhelmed by excessive exposure to reactive that not every liposomal vesicle may protect adequately the
oxygen species, a promising strategy for enhancing skin active agent. It depends on the structure, which can suffer
protection from oxidative stress would therefore be to support modifications when including the active agent.11 To date,
the endogenous skin antioxidant system. there has not been a proof of concept concerning the critical
Numerous antioxidants have been tested for their ability parameters in this respect and experimental data are needed
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
to reduce free radicals to less reactive molecules, thus mini- in order to substantiate a claim in this regard. Liposomes
mizing oxidative damage to critical cellular constituents.3 have been intensively investigated as carriers for different
The antioxidants for preventing skin oxidative damage applications in dermatology. The ability of some specific
that have been studied most intensively are vitamins E and types of liposomal vesicles to diffuse through the different
C, coenzyme Q10, and lipoic acid, referred to as “network layers of the skin has also been demonstrated allowing a suc-
antioxidants”, which work synergistically to regenerate one cessful treatment of different pathologies while restricting
another. Although glutathione, coenzyme Q10, and lipoic acid the systemic exposure to drugs.12
can be synthesized by humans, levels of vitamins C and E The present work evaluates the role of a specific ascorbate
depend on their oral intake or topical delivery.4 Vitamin C phosphatidylcholine (PC) liposome formulation as a carrier
is important in treating skin pathologies ranging from mild to improve the topical ascorbic acid treatment of skin. To
inflammation to skin cancer.5 this purpose, human skin penetration, inflammation, and
However, oral supplementation of vitamin C is not oxidation were evaluated.
thought to increase the skin concentration sufficiently.6
Materials and methods
For personal use only.
592 submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dermatology 2015:8
Dovepress
To prepare the fluorescein liposomes, we did as follows: Signed informed consent was obtained for each patient.
the lipid phase was done in the same way as in the sodium Ten Caucasian patients (40–50-years-old) free of any topical
ascorbate liposomes. Fluorescein was then dissolved in treatment gave the consent to use the leftover skin after plas-
apyrogenic and double-distilled water with the aid of sodium tic surgical abdominal resection procedure. Skin was used
hydroxide to form sodium fluorescein. The final concentra- for percutaneous absorption ex vivo assays. Skin was fatty
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
tion of fluorescein was 1 mg/mL. The two phases were mixed cleaned and frozen at -20ºC up to 3 months. The ex vivo stud-
and agitated with a food mixer. A solution of water and ies were performed in Franz diffusion cells using epidermis,
phenoxyethanol (conservative) was added to the liposome dermis, and whole skin as membranes. Epidermis and dermis
suspension. Liposome pH was between 9 and 8.5. A final were obtained using the method of Kligman.13 Briefly, it con-
pH of approximately 8.5 was achieved by adding a small sists of the separation of the upper layers (stratum corneum
amount of hydrochloric 1N. The suspension was incubated and viable epidermis) after the skin has been immersed in
at room temperature for 10 minutes and then filtered through distilled water at 60°C for 45 seconds.
the Minisart® syringe filter with 0.2 µm pore size.
Size determination, polydispersity index, and zeta poten- Franz diffusion cells system
tial were done with the Delsa Nano C Particle Analyzer The passive diffusion across human abdominal skin was
(Beckman Coulter Alcobendas, Madrid, Spain). The particle investigated using Franz diffusion jacketed cells with
size determination was performed by a method of dynamic flat grounded (ground O-ring) joints composed of clear
light scattering and the zeta potential by an electrophoretic borosilicate glass with an orifice diameter of 10 mm and a
For personal use only.
light scattering method based on the Doppler effect. The receptor volume of 5 mL (Franz cell apparatus, PermeGear
polydispersity index was used as a measure to describe the Inc., Hellertown, PA, USA). The Franz diffusion cells were
uniformity of the size distribution. A value of polydispersity mounted on a Franz cell stirrer with acrylic cell holders,
index equal or below to 0.12 indicates that the population is synchronously stirred using magnetic Teflon stir bars at a
homogenous. The zeta potential is used as the index of the constant speed of 500 rpm at 220 V/50 Hz (Franz cell stir-
surface charge of the particles. If the zeta potential is high rer, PermeGear Inc.), and thermostabilized. Skin sections
(30–150 mV), the liposomes are stable due to high electro- were mounted with the epidermal side of the skin (if pres-
static repulsion between particles and, on the contrary, a low ent) exposed to ambient conditions while dermal side was
zeta potential value increases the probability of particles bathed by phosphate-buffered saline (PBS) pH 7.4 at 36±1°C
colliding and thus forming particle aggregates. in the receptor medium. To achieve higher reproducibility,
The encapsulation efficiency (E%) was calculated using the skin sample was prehydrated with the receptor medium
the formula: E% = EA/TA × 100 where EA represents the for 60 minutes before applying the formulation. All bubbles
amount of encapsulated sodium ascorbate and TA the total were carefully removed between the underside of the skin
amount of sodium ascorbate initially added to solution. E% and solution in the receptor compartment.
in the liposomes was measured by determining the amount The liposomal formulations were applied onto the skin
of sodium ascorbate remaining after dialysis against distilled surface area of 0.9 cm2 in a nonoccluded condition. A mini-
water. Samples were injected into a dialysis cell (Slide-A- mum of three diffusion cells was used for each product. All
Lyzer® Dialysis Cassettes, Pierce, NJ, USA) with a hydro- studies were done in conditions of light protection of the
philic cellulose membrane (10,000 Molecular weight cut-off Franz cells and samples. After 20 hours, the experiments were
[MWCO]). After 4 hours of dialysis, the dialyzing medium stopped and the diffusion setup was dismantled.
was changed and left overnight.
After 24 hours of dialysis, samples were withdrawn for Biopsy processing for histology and
high performance liquid chromatography (HPLC) analysis staining
(the reference solution was a sample that was not dialyzed). At defined incubation times, whole skin samples were extracted
The experiment was repeated three times for each sample. from Franz cell and fixed in 4% buffered formaline pH 3.7
at 4°C for 24 hours. Then, a progressive OCT inclusion was
Human skin samples for permeation done using PBS in three steps for 1 hour each: OCT:PBS (1:3),
studies OCT:PBS (1:1), and OCT. Cryostat (Leica CM1900) at -15ºC
The study was approved by the Research Clinical Ethical was used to prepare 5 µm skin sections. Oil red stain was
Committee of the University General Hospital of Valencia. conducted in prewarmed Oil Red O solution for 8–10 minutes
Clinical, Cosmetic and Investigational Dermatology 2015:8 submit your manuscript | www.dovepress.com
593
Dovepress
in 60ºC oven and then Gill’s hematoxylin for 30 seconds. tissue samples, based on the scavenging of the 2,2′-azino-bis-(3-
An aqueous mounting medium for oil red stains and a specific ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical
fluorescent mounting medium (DAKO, Nottingham, UK) were (ABTS•), a soluble chromogen that is converted into a color-
used for optic and fluorescence microscopy, respectively. less product when it reacts with the ferryl myoglobin radical
(a product from the reaction between metmyoglobin and
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
Spain), and mobile phase, potassium hydrogen phosphate solar simulator (+UVA/UVB ranging 270–700 nm) with
buffer pH 2.6. The flow rate was 1.0 mL/min and the injec- an intensity of 50 J/cm2 for 2 hours with 1.7 mW/cm2 (Sun
tion volume was 100 µL. The HPLC method was validated Simulator UVA/UVBCUBE400; Hoenle system, München,
for accuracy (relative error obtained was ,10%), precision Germany) according to the OCDE 432 phototoxic test with
(2.7% as the highest variation coefficient), and linearity over modifications,14 whereas controls were kept in the dark. After
the concentration range analyzed (r2.0.996). The determina- another 24 hours of incubation in Franz cells, human skin
tion limit was 0.1 µg/mL. Concentration of ascorbate diffus- was retired and cut in small pieces and homogenized with
ing across the skin was expressed as µg/cm2 of skin. Ultra-Turrax (IKA, Staufen, Germany) in 500 µL of PBS.
Skin tissue solution was assayed for antioxidant capacity and
Fluorescein analysis and fluorescence for TNFα and IL-1β cytokines.
microscopy in skin permeation studies
Fluorescein liposomes skin absorption was analyzed by Statistics
collecting fluorescein samples directly from the receptor Statistical analysis was performed with the software program
medium of Franz cells, protected from light, and measuring GraphPad Prism 5. One-way ANOVA data analysis followed
the concentration by fluorometry with a plate spectropho- by the Bonferroni multiple comparison test was performed
tometer (PerkinElmer Inc., Waltham, MA, USA; victor 3). when appropriate on the permeability coefficients (Kp)
A standard curve was constructed with known concentra- and different groups of treatments to establish differences
tions of fluorescein to calculate fluorescein concentration between the calculated means.
as µg/cm2 of skin.
A Fluorescence Microscope Leica® DM6000B, equipped Results
with a photographic camera DFC480 and a Leica GFP filter, Analysis of liposome formulation and
was used to monitor fluorescein liposomes skin absorption. human skin penetration
Excitation of the sample was carried out with 470/40 nm blue All liposome formulations used in this study showed a lipo-
light. The fluorescent emission obtained was of 525/50 nm, some diameter in the range of 80–120 nm, with a polydisper-
in green light. sity index below 0.12 and a zeta potential between 30 and 150
mV. The final concentration of sodium ascorbate was 100±11
Skin antioxidant capacity, TNFα, mg/mL resulting in an E% of ∼40% (Table 1).
and IL-1β measures Whole human skin penetration assays were evaluated by
Total antioxidant status of the patients was estimated using a histology and measuring cumulative amounts of active agent.
Trolox™ Equivalent Antioxidant Capacity assay in human skin Oil red staining skin histology was performed to label lipids
594 submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dermatology 2015:8
Dovepress
Table 1 Physical characteristics of liposomes: z-average mean size and dermis (without stratum corneum and epidermis). As
(d), polydispersity index (PI), zeta potential (ζ), and encapsulation shown in Figure 3, whole skin penetration (samples collected
efficiency (E%) for sodium ascorbate
from the receptor medium of Franz cells) of fluorescein and
Formulation d (nm) PI ζ Potential (mV) E (%)
ascorbate, after corresponding liposomes applications, was
1 98.1±2.2 0.08±0.01 -76.4±1.8 38±1.1 small, reaching 8.94E-05±3.90E-06 kp (cm2/s) and 7.70E-
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
Clinical, Cosmetic and Investigational Dermatology 2015:8 submit your manuscript | www.dovepress.com
595
Dovepress
A B
15 min 30 min 1h
2.0 Liposomes
Solution
Fluorescein cumulative
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
amounts (µg/cm2)
1.5
3h 7h 20 h
1.0
0.5
30 h 70 h 70 h solution
0.0
0 20 40 60 80
Time (h)
tory cytokines. UVA/UVB exposure enhanced human skin of drug absorption into epidermis.15 On the other hand, dif-
For personal use only.
inflammation, increasing TNFα and IL-1β amounts in whole ferent advantages such as their ease of preparation, enhanced
human skin tissue. In contrast to ascorbate solution, ascor- absorption of active ingredients by skin, and continuous supply
bate liposomes pretreatment prevented the increase of TNFα of agents in a sustained period of time make these carriers also
and IL-1β induced by UVA/UVB irradiation (Figure 4B suitable for cosmetic applications.16 Our results showed that
and C) which is in agreement with the antioxidant results of PC liposomes are versatile as they can be used to encapsulate
ascorbate liposomes. both hydrophilic and lipophilic molecules. Sodium fluorescein
(hydrophilic, anionic) is a model of low lipophilicity active
Discussion agent and sodium ascorbate (unstable) as a hydrophilic mol-
In this study, we provide new evidence on topical bioavail- ecule. To be effective, an active drug must cross the stratum
ability of novel ascorbate liposomes, as well as their anti- corneum barrier. To achieve this, there are different factors
oxidant and anti-inflammatory properties, which protect skin to pay attention to: initial concentration of drug, diffusion
from UVA/UVB irradiation and may be of potential value coefficient, molecular size, partition coefficient, pH ioniza-
to attenuate different skin disorders. tion degree of the drug, anatomy of the stratum corneum, and
During recent years, the topical delivery of liposomes has vehicle–drug interactions. According to previous studies, the
been applied to different applications and in different disease hydrophobic liposome structure interacts with corneocytes
models. After topical application of liposomal formulations, and improves access to epidermis.17 Hydrophobic liposomes
such formulations can significantly increase the rate and extent allow the active component (ascorbate or fluorescein) to reach
A B
30 30
Epidermis
Fluorescein cumulative
Ascorbate cumulative
Dermis
amounts (µg/cm2)
amounts (µg/cm2)
20 20 Full skin
10 10
0 0
0 2 4 6 8 0 5 10 15 20 25
Time (h) Time (h)
Figure 3 Comparative skin penetration of fluorescein and ascorbate liposomes in different human skin layers.
Notes: (A) Fluorescein and (B) ascorbate liposomes penetration kinetics in epidermis (circle), dermis (square), and whole skin (triangle) of human abdomen. Experiments
were conducted in skin from four patients and run in triplicate. Results are expressed as mean ± standard deviation.
Abbreviation: h, hours.
596 submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dermatology 2015:8
Dovepress
the epidermis. However, it should be mentioned that the lipo- molecule of choice to achieve deeper targets such as the dermis
somes are loaded with active agent both inside and outside of and/or hypodermis (see Figure 3 in “Results” section).
their phospholipid membrane; therefore, the physicochemical Vitamin C, unique in its high reactivity with all aggressive
characteristics and mainly the n-octanol/water partition coef- oxygen radicals, is a major and the only essential antioxidant in
ficient (log Pow) of the active agent will also be factors that the aqueous cell compartment. Most animals synthesize their
influence the transepidermal absorption. In the case of the own vitamin C, but only humans and other primates lack the
target compounds, fluorescein is more lipophilic and has a enzyme α-glucono-γ-lactone oxidase, essential for vitamin C
higher molecular weight than ascorbate, which explains that synthesis. As active transport of vitamin C from the gastrointes-
the diffusion of ascorbate in the epidermis is lower with respect tinal tract is limited, even massive oral doses do not increase its
For personal use only.
to fluorescein due to the low lipophilicity of ascorbate. Once skin concentration to optimal levels.6 The skin contains relatively
dermis was reached, liposome diffusion was lower due to the low amounts of ascorbic acid, ∼41 ng/mg (dry weight) for the
higher hydrophilicity of this milieu and the degradation of the entire skin, with the stratum corneum containing only 7 ng/mg
ingredients by enzymatic means. Just the fraction released from (dry weight).18 Its level in the skin, especially in the epidermis,
the vesicles can penetrate easily through the dermis. These also decreases with age.19,20 Skin levels of vitamin C can also be
results were verified by comparing the absorption profiles of severely depleted by UV radiation and environmental pollutants.
the liposomal suspensions with aqueous solutions containing Even minimal UV exposure (a 1.6 minimal erythema dose)
the ingredients at the same concentrations than in dermis. decreases the level of vitamin C to 70% of the normal level
Therefore, once penetrated through the epidermis, the char- and exposure to 10 ppm of ozone in city pollution decreases its
acteristics of the active ingredient play a decisive role in the epidermal level by 55%.6 Topical application has been shown
degree of diffusion across the dermis. In this case, the ascorbate to increase significantly its cutaneous levels.
presents the hydrophilic characteristics suitable for getting In fact, we observed a significant increase of ascorbate
through this layer. Hence, although the liposomal fluorescein penetration with the PC liposome formulation in whole skin
has a faster profile to cross the epidermis, ascorbate is the as well as epidermis and dermis which may be of potential
A Control
Ascorbate liposome
Ascorbate solution
Empty liposome
B C
TNFα (ng/mg skin tissue)
# * * * *
1,500 600 *
Trolox (mM)
1.0 *
1,000 400
0.5 # #
500 200
* * *
0.0 0 0
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
U k
VA
ar
ar
ar
ar
ar
ar
ar
ar
ar
ar
ar
ar
D
Figure 4 Antioxidant and anti-inflammatory capacity of ascorbate liposomes in human skin irradiated by ultraviolet (UV)A/UVB.
Notes: Human whole abdomen skin was exposed to ascorbate solution or ascorbate liposomes (100 mg/mL) during 20 hours. After washing, skin surface was irradiated with
UVA/UVB intensity of 50 J/cm2 for 2 hours and remained incubated in Franz cells for 24 hours. Control preparations were left in Franz cells, in dark conditions. Skin tissue
was triturated and homogenized, and antioxidant Trolox capacity (A), tumor necrosis factor alpha (TNFα) (B), and interleukin (IL)-1β (C) were measured by antioxidant and
ELISA kits. Experiments were done in skin from four patients and run in triplicate. Results are expressed as mean ± standard deviation. *P,0.05 vs control dark conditions;
#
P,0.05 vs control group.
Clinical, Cosmetic and Investigational Dermatology 2015:8 submit your manuscript | www.dovepress.com
597
Dovepress
value to provide adequate antioxidant properties to the skin conditions can be different to those expected in human
at risk of oxidative or inflammatory damage. skin photodamage.
Photoaging is accelerated aging of the skin resulting
from environmental damage. The process is mainly due to Conclusion
UV irradiation and can be prevented. It accounts for most In summary, we showed a new ascorbate PC liposome for-
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
of the aging symptoms on sun-exposed skin. Knowledge of mulation with appropriate skin penetration, and antioxidant
the mechanisms involved in photoaging provides the key and anti-inflammatory properties against UVA/UVB pho-
approach for prevention and treatment of aged skin. In this todamage. Although the PC liposomes assayed in this work
regard, topical retinoids, particularly tretinoin, isotretinoin, showed promising results, other clinical assays are to be done
and tazarotene, are medical therapies with proven benefits to extract definitive conclusions.
for the treatment of photodamaged skin derived from ran-
domized clinical evidence.21 However, antioxidants have also Acknowledgments
been shown to have beneficial effects on reversing photoaged This work was supported by grants SAF2011-26443 (JC),
skin damage. Antioxidants are a very heterogeneous group FIS CP11/00293 (JM), FIS PI14/01733 (JM), CIBERES
of compounds varying from those similar to physiological (CB06/06/0027), ADE10/00020 (Spanish Government), as
species to numerous substances derived from nutritive plants, well as research grants from Regional Government Prometeo
particularly polyphenols, catechins, flavonoids, and acyclic II/2013/014 (JC, JM) “Generalitat Valenciana”. Sesderma
carotenoids, which also demonstrate strong antioxidant activ- laboratories partially support this work.
For personal use only.
598 submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dermatology 2015:8
Dovepress
11. Sharma VK, Sarwa KK, Mazumder B. Fluidity enhancement: a critical 17. Betz G, Imboden R, Imanidis G. Interaction of liposome formulations
factor for performance of liposomal transdermal drug delivery system. with human skin in vitro. Int J Pharm. 2001;229:117–129.
J Liposome Res. 2014;24:83–89. 18. Pugliese PT. The skin’s antioxidant systems. Dermatol Nurs. 1998;10:
12. Pierre MB, Dos Santos Miranda Costa I. Liposomal systems as drug 401–116:quiz 17–18.
delivery vehicles for dermal and transdermal applications. Arch 19. Leveque N, Muret P, Mary S, et al. Decrease in skin ascorbic acid
Dermatol Res. 2011;303:607–621. concentration with age. Eur J Dermatol. 2002;12:XXI–XXII.
13. Kligman AM, Christophers E. Preparation of isolated sheets of human 20. Leveque N, Robin S, Makki S, Muret P, Rougier A, Humbert P. Iron
Clinical, Cosmetic and Investigational Dermatology downloaded from https://www.dovepress.com/ by 110.139.106.16 on 13-Jan-2019
stratum corneum. Arch Dermatol. 1963;88:702–705. and ascorbic acid concentrations in human dermis with regard to age
14. Ceridono M, Tellner P, Bauer D, et al. The 3T3 neutral red uptake pho- and body sites. Gerontology. 2003;49:117–122.
totoxicity test: practical experience and implications for phototoxicity 21. Kohl E, Steinbauer J, Landthaler M, Szeimies RM. Skin ageing. J Eur
testing – the report of an ECVAM-EFPIA workshop. Regul Toxicol Acad Dermatol Venereol. 2011;25:873–884.
Pharmacol. 2012;63:480–488. 22. Marionnet AV, Chardonnet Y, Viac J, Schmitt. Differences in responses
15. Yoo J, Shanmugam S, Song CK, et al. Skin penetration and retention of of interleukin-1 and tumor necrosis factor alpha production and
L-ascorbic acid 2-phosphate using multilamellar vesicles. Arch Pharm secretion to cyclosporin-A and ultraviolet B-irradiation by normal and
Res. 2008;31:1652–1658. transformed keratinocyte cultures. Exp Dermatol. 1997;6:22–28.
16. Raj S, Jose S, Sumod US, Sabitha M. Nanotechnology in cosmetics:
opportunities and challenges. J Pharm Bioanal Sci. 2012;4:186–193.
For personal use only.
Clinical, Cosmetic and Investigational Dermatology 2015:8 submit your manuscript | www.dovepress.com
599
Dovepress