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J. Cosrnet. Sci.,
59,59-69 (JanuarylFebruary
2008)
Biological
activities of selected
peptides:
Skin
penetration
ability 0l copper complexes with
peptides
LENA MAZURO\(SKA and MIROSLA\7 MOJSKI, IVarsaw
Uniaersity of Tecbnologl, Faculty of Cheruistry, Noakoruskiego
3,
0
1
-664.W'arsaw,
Poland.
Accepted
for publication
August 15, 2007.
Synopsis
This study concerning the permeability through skin barriers of copper complexes with peptides is an
important part of the reseatch on theit biological activity. The transport of copper compiexes through the
skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements
in the skin. During the iast several years, a special interest in transepidermal copper delivety has been
observed. This is the reason why copper compounds have been used as active compounds in care cosmetics.
Yet, the transport process of copper complexes with ttipeptides, glycyl-histidyl-lysine GHK, or.y-glutamyl-
cysteinyl-glycine GSH thtough the stratum corneum has received very little attention in the literature so
far.
The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the
complexes with tripeptide on the coppet ion transport process is the key factor in their cosmetic and
pharmaceutical activity. The in uitro penetration process was studied in the model sysrem, a Franz diffusion
cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to
the ones ofthe intercellular cement ofstratum corneum were used as a standard model ofa skin barrier. The
results obtained demonstrated that coppet complexes permeate thtough the membranes modeling the horny
lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion.
INTRODUCTION
The existence of metal ions is essential
for all living organisms because
they are con-
stituents of a large group of enrymes responsible for different physiological processes.
Thus, they determine the proper functioning of the whole body, including the skin
tissue. One of these essential metals is copper, which, according to its antiradical
activity, the potential of regulating the melanogenesis
process, and the synthesis of
collagen, elastine, and GAGs (1,2), is widely used as a cosmetic ingredient.
Despite the fact that copper is one of the most important metals for normal skin activity
and growth, not all of the copper compounds, because
of their toxicity, may be used as
Address all corresoondence to Lena Mazurowska.
59
60 TOURNAL OF COSMETIC SCIENCE
cosmetic ingredients. Therefore, the simplest form of copper, an inorganic salt, cannot
be a possible source of delivery of the metal ions to the low layers
of skin because of its
general toxicity to the organism. This is why other ways of transporting copper to the
deep
layers of skin tissue have to be found. One of the widely used methods of delivering
metal ions into the skin is its complexation with different ligands, among which amino
acids and peptides play a main role.
In our investigation, small biological active peptides were used because, beyond their
rransporr
porential, they may function as an active ingredient in the cosmetic formulae.
Active peptides show cosmetically interesting activities such as stimulation of collagen
synthesis, chemotaxis,
and antistaining effects
(3).
Among many possible natural ligands, GHK and GSH are mainly used due to their
properries. Both peptides are intensively investigated because
of their existence in the
human organism and the different biological effects that they may show. The complexes
of GHK and GSH with copper are widely known as protection and repair agents for skin
tissue and because of this are often used as cosmetic ingredients.
Originally, GHK-Cu glycyl-histidyl-lysine-Cu(Il) was found in human plasma, and all
of its properries were drawn on the basis of these
investigations. GHK-Cu was isolated
from human plasma by Pickart and Thaler in 1973 (4). (see
also references
5 and 6).
Formeily, GHK peptides found an application in medicine. This peptide was first
described as a growth factor for avariety of differentiated cells. \fhat is more, recent data
suggest
its physiological role is related
to the process ofwound healing and tissue
repair
(7-10).
In further studies it was recognized that GHK is endowed with a wide range of more
systemic biological activities including angiogenesis
(blood vessel
formation) (11), ac-
celeration of bone repair (I2), and superoxide dismutase-like activity (Ir. GHK may
also have other activities when it is complexed with the Cu metal ion, like the secretion
of the tissue inhibitors of metalloproteinase
(14).
GSH is the next peptide that was isolated from a human body and enjoyed many
researchers' attention. The tripeptide 1-glutamyl-cysteinyl-glycine (GSH) is the maior
nonenzymatic regulator of intercellular redox homeostasis
and is ubiquitously present in
all cell types at millimolar concentrations. This cystein-containing tripeptide exists
either in a reduced (GSH) or oxidized (GSSG) form, better referred to as glutathione
disulfide, and participates in redox reactions by the reversible oxidation ofits active thiol
(15). Glutathione in the reduced (GSH) and oxidized (GSSG) forms is the main intra-
cellular non-protein thiol that performs the important biological functions involved in
active transport of amino acids (ry-glutamyl cycle), operating enzymes (glutathione
S-transferase, glutathione peroxidase, and glutathione reductase), complex formation
with microelements (Zn'*, Cu2*), and functioning of the redox couple Cu2*-Cu* (16).
GSH has many ascribed biological functions for skin, and one of them is implicated in
skin lightening. ln uiuo and in aitro studies in the literature show the evidence of its
involvement in the melanogenic pathway and shed light on its anti-melanogenic effect.
The proposed mechanisms of action include the direct inactivation of the enzyme ty-
rosinase by binding with the copper-containing active site of the enzyme and by me-
diating the switch mechanism from eumelanin to phaeomelanin production.
SKIN PENETRATION BY COPPER_PEPTIDE COMPLEXES ol
In the literature we may find that GSH has an activity of reducing free radicals and
peroxides that are responsible for tyrosinase activation, melanin formation, and modu-
lation of the depigmenting abilities of melanocytotoxic agents. This leads to the skin
lightening effect of GSH application and a possibility of its usage in the treatment of
pigmentary disorders (Il-20). Another important issue in skin protectioning is the
anti-UV (UVA and UVB) radiation activity of the cosmetic ingredient. Glutathione is
one of the ingredients that may play such a rcLe
(2I-24).
Due to the lack of data concerning the transport of the peptides and their complexes
through the skin, we focused our research on this subject. The main goals of our
experiments were to prove the ability of copper tripeptide complexes to penetrate the
skin, to determine the permeability coefficient
for these compounds, and to establish the
form of the compound that actually penetrates through the membrane. Our previous
study (25) proved that cooper
peptides can migrate through the model lipophilic mem-
brane from an aqueous solution (25), which made us continue the investigation of the
transport of copper peptide complexes, but this time through an emulsion.
Since most of the cosmetic formulae used as a source of active ingredients, like peptides
and their complexes, are O/Sf emulsions, we used them in our investigations. ihr' l,
uitro penetration process was studied in the model system, aFranz diffusion cell (26-28)
with a liposome membrane, where liquid crystalline systems of physicochemical prop-
erties similar to the ones of the intercellular cement of stratum corneum were used as a
standard model of a skin barrier (29-32).
MATERIALS AND METHODS
T\?ES OF APPARATUS
The absorption spectra were recorded using a SPECOL 11 spectrophotometer
(Zeiss,
Jena, Germany) with 5-mm glass
cells. The pFI measurements were carried out using an
Elmetron ES24 pH meter (Poland).
The reversed-phase liquid chromatographic experiment (RPLC) was performed by a
Perkin Elmer binary LC 250 computer-controlled
pump (Norwalk, CT) and a Rheodyne
model 7125 rnjection vial with injection loops (20 pl) (Cotati, Rheodyne, CA) with a
Perkin Elmer model LC-9, UV/Vis spectrophotometric detector. Peptides were sepa-
rated on the Hypersil BDS C1B analytical column (4.0 x I25 mm) (Agilent Technolo-
gies, $Tilmington, NC). The acquisition and handling of the data were carried out with
a IO20 LC PIus (Perkin Elmer) computer program. The copper
peptide complexes were
characterized
by an ESI mass spectrometer,
LC-MSD 1100 (Agilent) with a quadrupole
mass analyzer (HP75004).
REAGENTS
o A stock Cu(II) solution, (1 mg/ml-1) was obtained by dissolution of copper(Il) chlo-
ride dehydrate (POCH, Gliwice, Poland) in water.
o A GHK-Cu solution (0.01 M) was
prepared by dissolution of Prezatide copper acetate
(GHK-Cu) (ProCyte Corporation, USA) in water.
o A GSH stock solution (lOmg/ml) was
prepared
by dissolution of glutathione (Sigma-
oz
o
a
JOURNAL OF COSMETIC SCIENCE
Aldrich) in water.
The solution was diluted in a calibrated
flask. The complex of GSH
with Cu was molar ratio 2'.I and 1:1.
A buffer solution (pH 1
.4) was prepared by dissolving potassium phosphate
(POCH,
Gliwice, Poland), and its pH was adjusted to 7
.4 by addition of di-sodium hydrogen
phosphate dodecahydrate
(POCH, Gliwice, Poland). The obtained solution was di-
luted to 1000 ml with demineralized water.
A O.I% biscyclohexanon-oxalyldihydrazone
(cuprizon) (Fluka, Buchs, Switzerland)
solution was
prepared by dissolving 2OO
mg of cuprizon in 40 ml of hot 50% ethanol.
This solution was diluted with ethanol to 200 ml.
A buffer solution (pH 10.0) was
prepared by dissolving ammonium chloride (POCH,
Gliwice, Poland) and was adjusted to 10.0 by addition of ammonium (POCH, Gli-
wice, Poland). The obtained solution was diluted to 1000 ml with demineralized
watef .
Trifluoroacetic acid (TFA) soluti on (0.I5%o) (Fluka, Buchs, Switzerland) was
prepared
by dissolving an appropriate amount in distilled water. The solution was diluted in
a calibrated flask.
Components qf the model emulsion: 8% glyceryI stearate (Cutina GMS); 20%
hexyldecanol, hexyldecyl laurate (Cetiol PGL); )% emulsifier-Ceteareth-20 (Eumul-
gin B2); O.I% methylchloroisothiazolinone,
methylisothiazolinone (Kathon CG); and
watef-q.s.
PREPARATION OF THE MEMBRANE
The lipophilic membrane for modeling stratum corneum lipids was prepared by sand-
wiching O.I25 mI of liposomes (Cerasome)
(Lipoid GmbH, Germany) composed of the
horny layer lipids. The appropriately thick lipid layer was placed between two mem-
branes
(Institute of Chemistry and Nuclear Technique, Poland) of polyester foil (radius,
T2 mrr' diameter of pores,
0.4 micrometer; thickness, 12 micrometers). The membrane
was left for 24 hours to evaDorate the water.
EXPERIMENTAL
ln uitro
membrane permearion experiments
were performed using aFranz diffusion cell.
The acceptor cell was filled with 15 ml of phosphate buffer (pH 1.4). One gram o{ a
O/.W emulsion containing copper complexes
with pept'l"les was
placed in the donor cell.
The available diffusion area
between cells was I.77 crn". The contents of the cells were
stirred at 1000 rpm by amagnedc stirrer.
During the-72
hours of experiments, the water
from the emulsion was evaporated. The experiments were conducted at room tempera-
tufe.
Copper was determined spectrophotometrically
at 600 nm. One milliliter of the solution
from the acceptor
cell (during 72 hours) was transferred
into a 10-ml calibrated flask,
and 2 ml of O.L% cuprizon and 2 ml of buffer solution (pH 10.0) were added. The
mixture was diluted to 10 ml inacalibration flask, and the absorbance
of the solution
at 600 nm against a reageflt blank was measured (33).
The determinarion of the total amount of tripeptide in the acceptor
cell was carried out
by RPLC. A 1-ml sample was carried out from the acceptor
cell. A 20-pl portion of this
sample was injected onto the column. The flow rate of the eluent (0.I5% TFA) was 0.7
a
SKIN PENETRATION BY COPPER-PEPTIDE COMPLEXES 63
ml/min-1, and the eluate was monitored at 2OO nm using the UV/Vis detector. The
concentration of peptide was determined by measuring the peak area.
Electrospray MS was applied to identiĄ' copper complexes
present in the acceptor
cell.
ESI-MS spectra were acquired in the range of 150-1500 p using 20 ms dwell time and
0.1 p of step size.
The ion spray voltage of 4OO0
V was applied for positive and negative
ion acquisition. The orifice potential was established
at 80 V, as the one offering the best
signal intensity and causing partial fragmentation of the molecular ion at the peptide
bounds (34).
DATA ANAIYSIS
To calculate the permeability coefficient, the cumulative amount of copper ions was
plotted against the flux Q) of a compound across the membrane, determined at steady
state (35). The permeability coefficient of the Cu2* ion in the lipid membrane Ko
(cm's 1) was calculated by Fick's first law of diffusion. Figure 1 shows exemplary
permeation profiles of ligands and the amount of copper vs time.
RESULTS AND DISCUSSION
The aim of our research was to determine the influence of ligands (peptides) on the
permeation process
of copper ions. First, our studies
confirmed the ability of copper ions
to penetrate the model membrane without the determination of a compound form
(copper
ions or copper complexes). Second, we investigated
the concentration
ofpeptides
that permeated the membrane. Finally, from the obtained data and ESI-MS results we
were able to establish the form in which copper and peptides permeate.
PERMEABILITY COEFFICIENT STUDY
The results introduced in Figure 2 reveal a high influence of complexing agent
(GHK
or GSH) on the permeability coefficient of copper ions. In all cases,
the permeation rates
of copper ions were lower than those obtained for complexed copper. For this reason, it
may be concluded that the complexing agents (GHK and GSH) accelerate
the migration
of copper ions through the model membranes. As shown in Figure 2, the influence of
peptide complexes on the permeation of copper ions has different levels;
the influence of
GHK on copper ion penetration was confirmed to be twice as strong as that of GSH.
Research determined the permeation coefficient of peptides from the copper complexes.
The concentration of GHK and GSH in the acceptor
cell was determined by reversed-
phase liquid chromatography
(RPLC) with UV-VIS detection.
In Figure 3 the compari-
son of the permeation coefficients of GHK and GSH peptides from the copper complexes
is presented. The figure proves that GHK and GHK-Cu have very similar values. \fhat
is more, on the basis of Figure 3 the conclusion that tripeptide complexation of copper
does not change the Ko value of GHK may be drawn. The GSH values were different:
the Ko for GSH was higher rhan that for the GSH copper complex. Similar properties
of the-penetration abilities of the GHK peptides confirmed the thesis
that the structure
and high affinity to the lipid structures of the membrane strongly influence the per-
meation process. The permeation coefficients
of the peptides are significantly lower than
those of copper ions.
64
a)
JOURNAL OF COSMETIC SCIENCE
"1. *{. ,{-
,.'+
|.
08162432404856647280 time
[h]
}t' }ł{
081624324048566/.72 time
[h]
Figure 1. The permeation profiIes of peptide GSH from (a) copper complex GSH-Cu and (b) peptide.
ESI-MS STUDY
The other significant factor playin g a key role in the migration processes is the equi-
librium of the complexes in the acceptor solution. Finally, from the obtained data and
ESI-MS results, we were able to establish the form in which copper and the peptides
permeate. As shown in Figure 2, we could find a lot of molecules in the acceptor
cell by
ESI-MS study. During the study of the penetration ability of peptides we could find
species
in acceptor cells (GHK and GSH).
CD
{
;
At
T'
o
E
o
E
o
ą
2z
c
5
o
E
o
b)
6
;3
a
o
It
o
(E
E2
c
o
ą
b
c
e1
E
as
,o
E
o
o
.L
Y
SKIN PENETRATION BY COPPER_PEPTIDE COMPLEXES o)
2,00
1,50
1,00
0,50
0,00 Cu2+ GSH€u GHK€u
Figure 2. The influence
of peptide complexes
on the permeability of copper ions
0,o20
0,015
,o
E
-t o,olo
o
CL
Y0,005
0,mo
Figure 3. The GHK€u GHK GSH4u GsH
influence of complexation
on the permeation coefficients
of peptides
GHK and GSH.
The tripeptide GHK was identified by a mass spectrum, in which a signal at n/z 339
was registered and identified as a quasi-molecular ion of GHK (Figure 4a). A signal for
GSH compound was registered at m/z 306 (Figure 4b). ESI-MS study proves that
tripeptides GHK and GSH penetrate from the emulsion through the modeling stratum
corneum membrane (Table I).
The GHK-Cu complex from the emulsion penetrates
in different forms, like tripeptide
GHK and GHK-Cu (Figure 5a).
The mass spectrum for these studies consists of two
signals at m/z 339 for GHK and 4OO for GHK-Cu. These results
are similar to those in
our investigations for aquae's solution (25) atd show the influence ofthe structure ofthe
complexes on the penetration ability of the copper tripeptide complexes. This can
suggesr
that GHK-Cu is the mosr important species
in the penetration
ability of copper
complexes with GHK. It can be confirmed by the fact that the permeability coefficient
Ko for copper from the GHK-Cu complex is higher than that for copper ions alone.
A1l
the signals were compared to the theoretical profiles.
The ESI-MS results for the penetration ability of the GSH-Cu complex show that the
compounds that penetrate through the membrane are GSH and a very small amount of
66
a)
JOURNAL OF COSMETIC SCIENCE
b)
tm
80
60
%
/to
20
o
200 /tOO
"'- 20o
Figure 4. Mass spectra for (a) GHK and (b) GSH ftom emulsion in acceptor cells.
Table I
Ionized Species Observed
in ESI-MS Spectta
of GHK-Cu and
GSH-Cu Solutions
Positive ion Negative ion
Proposed ion Proposed ion
80
60
%
40
0
I
I
I
I
I
I
d
jrh
m/z
339
400
304
)o/
34r
402
306
369
IGHK + Hlt
ICuGHK +
Hl-
TGSH - H]-
TGSH _ H]-
IGHK + Hl-
ICuGHK - Hl
IGSH - Hl
IGSH - Hl-
GSH-Cu (Figure 5b). The mass spectrum for these studies consists of two signals at m/z
306 for GSH and 369 for GSH-Cu. The participation of the GSH ligand form in the
penetration ability of copper shows the important role of these
species in the transport
process. The ligand influence can be confirmed by the fact that the Ko for copper from
these
complexes is lower than for copper from the GHK-Cu complex. All signals were
compared to the theoretical profiles. This result supports the thesis that copper com-
plexes with bioligands can be formed and penetrate through the model membrane from
the emulsion.
CONCLUSIONS
The biological activities of the copper tripeptide complex play an important role in the
protection and regeneration
of skin tissue, and GHK-Cu and GSH-Cu are a very good
copper ion source. The research on copper transport through membrane modeling stra-
tum corneum proved that the tripeptide-copper complex may permeate through a horny
Iayer
of epidermis.
GHK significantly participates in the wound-healing process:
due to its properties it
influences the elasticity and strength of the skin. rilfhat's more, GSH play a very
x
.r,i.t
t--'-L i-,r,
SKIN PENETRATION BY COPPER-PEPTIDE COMPLEXES b/
a)1m
&]
60
%
40
o
b)
100
80
60
%
Ę
0
Figure 5. t^.rlp...ru f". (")
GHK:;"nd (b)
GSH-Cu from emulsion in acceptor cells.
important role in skin lightening. The research
proves that the copper complexes might
be a good source
nor only for copper ions but also for peptides.
The investigations ofthe
influence of complexing agents
on the skin migration rate of copper ions have
evidenced
their hampering role in this process.
At the presenr time, the incorporation of copper ions in cosmetics formulations is still
very popular in Europe. The benefici
al effect
of the active substances depends strictly on
their skin penerration ability. Our investigations show the possibility of the effective
penetration of copper complexes with tripeptide into the stratum corneum, which allows
one to exploit the biological activity of these complexes in cosmetics.
ACKNO\TLEDGMENTS
The authors are thankful to Lipoid GmBH (Germany) for the kind gift of Cerasome
9005 and to ProCyte (USA) for the sample of Prezatitde copper acetate. The authors are
grateful to Rafal Ruzik for valuable help with the ESI-MS study.
RTFERENCES
(1) R. H. G"y, J.J. Hostynek, R. S. Hinz, and C. R. Lorence, Metals and the Skin (Marcel Dekket, New
Yotu, I99), pp. 179-189.
(2) M. C. Linder, Copper and genomic stability in mammals, Mut' Res.,
475,I4I-I12 (2O0I).
(3) K. Lintner and O. Peschard, Biologicaily active peptides: From a laboratory bench curiosity to a
functional skin care product, lnt. J. Cosnet. Sci., 22,207-218 (2000).
(4) L. Pickart and M. M. Thaler, Tripeptide in human serum which prolongs survival of notmal liver cells
and stimulates growth in neoplastic livet, Nature Neu,, Biol., 243,8r-87 (I973).
(5) S.J. Lau and B. Sarkar, The interaction of copper(Il) and glycyl-L-histidyl-L-lysine, a growth-
modulating tripeptide from plasma, BiochenJ., 199, 649-616 (1981).
(6) C. Conato, R. Gavioli, R. Guerrini, H. Kozlowski, P. MIynarz, C. Pasti, F. Pulidori, and M. Remelli,
Copper complexes ofglycyl,histidyl-lysine and two ofits synthetic analogues: Chemical behaviour and
biological activity, Biocbim. Biopbys. Acta, 1526, I99-2lO (2O0I).
(7) F. X. Maquart, L. Pickart, M. Laurent, P. Gillery, J. C. Monboisse, and J. P. Borel, Stimulation of
E
I
l(
Iź
9.
ó
o
68 JOURNAL OF COSMETIC SCIENCE
collagen synthesis in fibtoblast cultutes by the ttipeptide-copper complex glycl-L-histidyl-L-lysine-
Co2*, FEB, 238,343-346 (1988).
(8) A. Simeon, J. S7egrowski, Y. Bontemps, and F. X. Maquart, Expression of glycosaminoglycans and
smail proteoglycans in wounds: Modulation by the tripeptide-copper complex glycyl-L-histidyl-L-
Iysine-Cu2*,,/. lnaest. Dermatol., 1I5, 962-968 (2000).
(9) L. Pickart, Biologl of Copper Conplexes (Humana Ptess, Clifton, New Jersey, 1987), pp. 213.
(10) D. Counts, E. HiIl, M. Turner-Beatty, M. Grotewiel, S. Fosha-Thomas, and L. Pickart, Effect of Iamin
on full thickness wound healing, FASEB J., 6, A1636 (1992).
(11) P. M. Gullino, Microenvironment and angiogenic response, EXS,6I, I25-I28 (1992).
(I2) H. Pohunkova, J. Stehlik, J. Vachal, O. Cech, and M. Adam, Morphoiogical featutes of bone healing
under the effect of collagen-gtaft-glycosaminoglycan copolymer supplemented with the tripeptide
Gly-His-Lys, B iomataials, I7, I5 67
-Ir7 4 (1996).
(13) N. Cotelle, E. Tremolieres, J. L. Bernier, J. P. Catteau, and J. P. Henichart, Redox chemistry of
complexes of nickel(Il) with some biologically important peptides in the presence of reduced oxygen
species: An ESR study,,/. Inorg. Biochern., 46,7-I5, (1992).
(I4) A. Simeon, H. Emonard, W. Hornebeck, and F. X. Maquart, The tripeptide-copper complex glycyl-
L-histidyl-LJysine-Cu2* stimulates matrix metalloproteinase-2 expression by fibroblast cul,tves, Life
Sci., 67, 2217
-2265 (2000).
(1t) A. Meister and M. E. Anderson, Glutathione, Annu. Rea, Biocben.,52,71I-760 (198r.
(16) V. G. Shtyrlin, Y.I. Zyavkina, V. S. Ilakin, R. R. Garipov, and A. V. Zakharov, Structure, stability,
and ligand exchange of copper(Il) complexes with oxidized glutathione,,/. Inorg' Bźochem.,
99, 133,_
1346
Q005).
(17) C. D. Villarama and H. L Maibach, Glutathione as a depigmenting agent: An overview, Int.
J. Cosrnet.
Sci.,
27, 147-153
Q00r).
(18) T. Yamamura, J. Onishi, and T. Nishiyama, Antimelanogenic activity of hydrocoumarins in cultured
normal human melanocytes by stimulating intracellular glutathione synthesis, Arcb, De'rnłłtol' Res',
294,349-359
Q002).
(19) G. Imokawa, Analysis of initial melanogenesis including tyrosinase transfet and melanosome differ-
entiation though interrupted melanization by glutathione,J. Ina. Detmatol.,93, 100-107 (1989).
(20) B. Kasraee, F. Handjani, and F. S. Aslani, Enhancement of the depigmenting effect of hydroquinone
and 4-hydroxyanisole by all-trans-retinoic acid (Ttetinoin): The impairment ofglutathione-dependent
cytoprotectiofl ?, Dernatology, 206, 289-29I (2003).
(21) M. A. Pelissier, N. Savoute, G. Briands, and R. Albtecht, Endogenous glutathione as potential pro-
tectant against free radicals in the skin of vitamin A deficient mice, Food Chem. Toxicol., 35,693-696
(1997).
(22) G. F. Vile and R. M. Tyrrel, UVA radiation-induced oxidative damage to lipids andprotetns in uitro
and in human skin fibroblasts is dependent on iron and singlet oxygen, Fru Rad. Bio!. Med., 18,
72r-730
(199r).
(23) R. Maselia, R. Bededetto, R. Vari, C. Filesi, and C. Giovannini, Novel mechanisms of natural
antioxidant compounds in biological systems: Involvement of glutathione and glutathione-related
enzymes,,L Nut. Bio., 16, 177-586 (2O0r).
(24) S. E. Tobi, N. Paul, and T. J. McMillan, Glutathione modulates the 1evel of free radicals produced in
UVA-irradiated ceIIs,J. Photocbem. Pbotobiol. 8., 57, I02-II2 (2000).
(25) L. Mazvowska and M. Mojski, ESI-MS study of the mechanism of glycylJ-histidyl-l-lysine-Cu(Il)
complex transport through model membrane of stratum corneum, Talanta, 72, 650-654 (2007).
(26) I.J. Bosman, A. L. Lawant, S. R. Aegaart, K. Ensing, and R. A. de Zeeuw, Novel diffusion cellfor in
uitro transdermal permeation, compatible with automated dynamic sampling,rl Pbarn, Biomd. AnaJ.,
M, rcr5-r023
Ggg6\
(27) J. Shokri, A. Nokhodchi, and A. Dashbolaghi, The effect of surfactants on the skin penerration of
diazepam, Int. J, Pbann., 228,99-107 (200I).
(28) A. Oborska, J. Arct, M. Mojski, and E. Jaremko, Influence of polyalcohols and surfactants on skin
penetration of flavonoids from the emulsion,,/. Appl. Cosmetol, 22, 3142 (2OO4).
(29) K. Matsuzaki, T. Imaoka, M. Asano, and K. Miyajima, Development of a model membrane system
using stratum corneum lipids for estimation of drug skin permeabiltty, Chern. Pharn. Bull., 4I,
J75-579
G993).
(30) J. Houk and R. H. Guy, Membrane models for skin penetration studies, Chem, Reu., 88,4)541I
(1988).
SKIN PENETRATION BY COPPER_PEPTIDE COMPLEXES 69
(31) M. Ricci, C. Puglia, F. Bonina, C. Di Giovanni, S. Giovagnoli, and C. Rossi, Evaluation of indo-
methacin percutaneous absorption from nanostructured lipid catriers (NLC): 1z uiło and in uiuo satdies,
J. Pharm. Sci.,
94, 1149-1.159 Q00t).
ę2) G, M. M' E. Maghraby, M. Campbell, and B. C. Finnin, Mechanisms of action of novel skin penetra-
tion enhancers: Phospholipid versus skin lipid liposomes, lnt. J. Pharn', 3O5,90-104 (20Or).
ę, L.]' A. Haywood and P. Sutcliffe' Determination of copper in steel, Analyst, 81,6'I_6,, (19,6).
ę4) K. Poleć-Pawlak, R. Ruzik' K. Abramski' M. Ciurzyńska, and H. Gawrońska, Cadmium speciation in
Arabidopsis tbaliana as a strategy to study metal accumulation system in plants, Anal. Cbim. Acta, 54O,
61-70
Q001\.
(35) H. Sćhaefer and T. Redelmeiet, Skin Barrier (Katger AG' Basel' 199ó).