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Biological activities of selected peptides: Skin penetration ability of copper complexes with peptides

Authors:

Abstract

This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery 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 tripeptides, glycyl-histidyl-lysine GHK, or gamma-glutamyl-cysteinyl-glycine GSH through 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 copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion.
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
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.
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... The use of Kp is now encouraged [22] although it has been criticized [23]. The increased uptake of Cu(II) in the presence of amino acids is in agreement with previous results obtained with Ehrlich ascites tumor, brain, liver and kidney cells [24][25][26]. The K p of Cu(II) complexed to alanine (6.31 ± 0.01) × 10 −6 cm/s and glycine (5.79 ± 0.04) × 10 −6 cm/s are comparable to values found by Mazurowsky for the same complexes (alanine (1.90 ± 0.16) × 10 −6 cm/s; glycine (1.62 ± 0.06) × 10 −6 cm/s) [24][25][26]. ...
... The increased uptake of Cu(II) in the presence of amino acids is in agreement with previous results obtained with Ehrlich ascites tumor, brain, liver and kidney cells [24][25][26]. The K p of Cu(II) complexed to alanine (6.31 ± 0.01) × 10 −6 cm/s and glycine (5.79 ± 0.04) × 10 −6 cm/s are comparable to values found by Mazurowsky for the same complexes (alanine (1.90 ± 0.16) × 10 −6 cm/s; glycine (1.62 ± 0.06) × 10 −6 cm/s) [24][25][26]. Mazurowsky used liposomes as a model membrane, with potassium phosphate buffered at pH 7.4 as the acceptor phase. ...
... The EF ranged from 2 for dtpa to 6.8 for H(555N) ( Table 1). The increased uptake of Cu(II) in the presence of amino acids is in agreement with previous results obtained with Ehrlich ascites tumor, brain, liver and kidney cells [24][25][26]. The Kp of Cu(II) complexed to alanine (6.31 ± 0.01) × 10 −6 cm/s and glycine (5.79 ± 0.04) × 10 −6 cm/s are comparable to values found by Mazurowsky for the same complexes (alanine (1.90 ± 0.16) × 10 −6 cm/s; glycine (1.62 ± 0.06) × 10 −6 cm/s) [24][25][26]. ...
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Membrane permeability of copper complexes with potential anti-inflammatory activity were measured using an artificial membrane in a modified Franz cell. Using CuCl 2 as the control, all the ligands tested enhanced the diffusion of copper, with enhancement factors ranging from 2 to 7. Octanol/water partition coefficients (log K o/w) were measured and correlated with the permeability coefficients (K p). In addition, chemical speciation was used to determine the predominant complex in solution at physiological pH. No correlation was found between the measured permeability coefficients and either molecular weight (MW) or log K o/w .
... В настоящее время для ряда регуляторных пептидов установлено влияние на процессы роста и дифференцировки клеток [3]. В частности трипептид глицил-гистидил-лизин NH 2 -Gly-L-His-L-Lys-COOH (GHK) обладает антиоксидантными, им-мунотропными, противовоспалительными и нейротропными эффектами [4][5][6][7], а также действует на процессы регенерации ткани [8][9][10]. Однако наряду с целым рядом достоинств у препаратов на основе регуляторных пептидов (высокая эффективность в сравнительно низких дозах, отсутствие значительных побочных эффектов) основным их недостатком является быстрая деградация протеолитическими ферментами. ...
... Также известно, что GHK при взаимодействии с ионами меди образует комплекс Gly-His-Lys-Cu (GHK-Cu), который активирует процессы пролиферации при раневых процессах в коже и положительный хемотаксис макрофагов в область повреждения [4,21]. Кроме того, общеизвестным фактом является участие двухвалентных катионов меди в образовании гидроксильных радикалов, обладающих высокой активностью и играющих важную роль в кислородзависимых бактерицидных механизмах фагоцитирующих клеток. ...
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Objective: to study the effects of the GHK peptide and its structure analogues on the mechanisms of innate immunity and lipid peroxidation in wound. Materials and methods. The experiments were performed on 70 male Wistar rats. The skin wound was modeled by applying a full-layer wound with an area of 250 mm2 on the animal's back. The peptides Gly-His-Lys (GHK), D-Ala-Gly-His-Lys (D-Ala-GHK), and Gly-His-Lys-D-Ala (GHK-D-Ala) were used in doses of 0.5 μg/kg and 1.5 μg/kg, which it was administered intradermally in doses of 0.5 μg/kg and 1.5 μg/kg in a volume of 0.1 ml for 3, 7 or 10 days. The activity of lipid peroxidation (LPO) processes in blood serum was assessed by the content of malonic dialdehyde (MDA) and acylhydroperoxides (AHP). The antioxidant mechanisms were evaluated by determining the activity of catalase and the total antioxidant activity (OAA) of blood serum. Phagocytic activity of blood neutrophils was assessed by phagocytic index (PI) and phagocytic number (PN). The activity of oxygen-dependent mechanisms in phagocytes was evaluated in a spontaneous nitroblue tetrazolium test (NBT). Results. After the administration of GHK, the tendency to PI, PN and completeness of phagocytosis prevailed, which mainly persisted with the use of peptides D-Ala-GHK and GHK-D-Ala. At the same time, the GHK-D-Ala peptide at a dose of 1.5 μg/kg had the most stable effect on phagocytic activity. The data obtained in the NBT are largely consistent with the PN indicators. At the same time, the effects of structural analogues, unlike GHK, were manifested throughout the experiment. Significantly significant changes in the activity of LPO and antioxidant mechanisms were observed with the use of all peptides. However, their dynamics, orientation and severity throughout the experiment were quite complex. Conclusion. GHK and its structural analogues, D-Ala-GHK and GHK-D-Ala, had an effect on the indicators of innate immunity and LPO in a skin wound, the severity and direction of which depends on the healing period. At the same time, the most pronounced and stable effects were observed when using GHK-D-Ala. That demonstrates the importance of protecting the tripeptide molecule from the action of carboxypeptidases.
... Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules30010136/s1, Figure S1: Structure of the Strat-M ® membrane; Table S1: Selected permeation tests; Table S2: Different skins comparable to Strat-M ® in terms of drug penetration ability; Table S3: Skin permeation studies of GHK-Cu; Table S4: Studies on skin permeation induced by liposomal encapsulation [82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100]. ...
Article
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Cosmetically active compounds (CACs), both of lipophilic and hydrophilic origin, have difficulty reaching the deeper layers of the skin, and this shortcoming significantly reduces their efficacy. One such CAC that occurs naturally in the human body and displays many beneficial properties (via reducing fine lines and wrinkles, tightening skin, improving its elasticity, etc.) is the glycyl-L-histidyl-L-lysine tripeptide complex of copper (GHK–Cu). GHK–Cu is a fairly hydrophilic compound with limited permeation through the lipophilic stratum corneum. On the other hand, liposomes capable of encapsulating GHK–Cu may improve its permeation potential. The present review discusses various issues related to obtaining insight into the permeation of CACs through the skin. Methods for studying the transport of CACs encapsulated by liposomes and free GHK–Cu across the skin barrier are summarized. An analysis of the literature data reveals that the transport of liposomes containing GHK–Cu received little attention. This research gap gives an impetus to the methodological developments for assessing the effect of liposomes on GHK–Cu transportation and trafficking.
... Interestingly, peptides are also useful in cosmetics and can be used as active ingredients on sensitive skin due to their ability to interact with skin cells with high potency at low dosage and to penetrate the stratum corneum [14]. ...
Article
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Numerous applications of amino acid-based compounds and peptide derivatives in different biomedicine- and nanotechnology-related fields were described in the recent scientific literature [...]
... Copper tripeptide is capable of skin penetration and acting as a delivery system for metal cations, and it can be used in patch technology for anti-inflammatory therapy. GHK peptide as a complexing agent for copper ions increases the permeation rates of Cu 2+ , presumably accelerating the migration of copper ions through lipophilic stratum corneum [11]. GHK-Cu is known for lowering the healing time of damaged skin; it is capable of modulating matrix metalloproteinases expressions that are crucial for the healing process [12]. ...
Article
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Liposomes are self-assembled spherical systems composed of amphiphilic phospholipids. They can be used as carriers of both hydrophobic and hydrophilic substances, such as the anti-aging and wound-healing copper-binding peptide, GHK-Cu (glycyl-L-histidyl-L-lysine). Anionic (AL) and cationic (CL) hydrogenated lecithin-based liposomes were obtained as GHK-Cu skin delivery systems using the thin-film hydration method combined with freeze-thaw cycles and the extrusion process. The influence of total lipid content, lipid composition and GHK-Cu concentration on the physicochemical properties of liposomes was studied. The lipid bilayer fluidity and the peptide encapsulation efficiency (EE) were also determined. Moreover, in vitro assays of tyrosinase and elastase inhibition were performed. Stable GHK-Cu-loaded liposome systems of small sizes (approx. 100 nm) were obtained. The bilayer fluidity was higher in the case of cationic liposomes. As the best carriers, 25 mg/cm 3 CL and AL hydrated with 0.5 mg/cm 3 GHK-Cu were selected with EE of 31.7 ± 0.9% and 20.0 ± 2.8%, respectively. The obtained results confirmed that the liposomes can be used as carriers for biomimetic peptides such as copper-binding peptide and that the GHK-Cu did not significantly affect the tyrosinase activity but led to 48.90 ± 2.50% elastase inhibition, thus reducing the rate of elastin degeneration and supporting the structural integrity of the skin.
... Several copper peptide complexes have been shown to calm irritated skin, improve elasticity, firmness, and accelerate wound healing [36]. Other copper compounds have shown antibacterial, anti-fungal, and antiviral properties [37]. ...
Article
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Introduction: The science in aesthetic medicine continues to grow and expand exponentially. With improved science comes advanced clinical therapeutics that further benefits our patients. Physicians are seeking non-surgical technologies that are capable of safely treating all Fitzpatrick skin types while yielding meticulous results.
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The increasing demand for effective cosmetics has driven the development of innovative analytical techniques to ensure product quality. This work presents the development and validation of a zwitterionic hydrophilic interaction liquid chromatography method, coupled with ultraviolet detection, for the quantification of copper tripeptide in cosmetics. A novel protocol for sample preparation was developed using fabric phase sorptive extraction to extract the targeted analyte from the complex cosmetic cream matrix, followed by chromatographic separation on a ZIC®-pHILIC analytical column. A thorough investigation of the chromatographic behavior of the copper tripeptide on the HILIC column was performed during method development. The mobile phase consisted of 133 mM ammonium formate (pH 9, adjusted with ammonium hydroxide) and acetonitrile at a 40:60 (v/v) ratio, with a flow rate of 0.2 mL/min. A design of experiments (DOE) approach allowed precise adjustments to various factors influencing the extraction process, leading to the optimization of the fabric phase sorptive extraction protocol for copper tripeptide analysis. The method demonstrated excellent linearity over a concentration range of 0.002 to 0.005% w/w for copper tripeptide, with a correlation coefficient exceeding 0.998. The limits of detection and quantitation were 5.3 × 10−4% w/w and 2.0 × 10−3% w/w, respectively. The selectivity of the method was verified through successful separation of copper tripeptide from other cream components within 10 min, establishing its suitability for high-throughput quality control of cosmetic formulations.
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It has been proven that common cosmetic ingredients - polyalcohols and surfactants can influence the permeation process of flavonoids from O/W model emulsion. The experiments have shown that permeation rate of selected flavonoids decreases with increasing length of surfactant's oxyethylene chain. The highest permeability coefficient was calculated for the emulsion containing polyoxyethylene 12 cetostearyl ether. It has been documented that investigated polyalcohols increase permeation rate of both quercetin and rutin. The substance increasing permeability coefficients of flavonoids very effectively is 1,2-propylene glycol. Investigations introduced in this paper have evidenced that permeation coefficients of flavonoids increase with increasing molecular weight of polyethylene glycols up to the value 1500. It has been also confirmed that the penetration ability of flavonoids depends on concentration of applied cosmetic additives. Results presented in this paper through a new light on the problem of formulating products containing flavonoids and posses many practical aspects. To elucidate the mechanisms that are involved in the processes described above the investigation on the model solution should be done. It has been proven that process of skin penetration of flavonoids can be controlled by proper selection of other cosmetic additives. The careful addition of adequate hydrophilic substance and application of suitable surfactant can increase the permeation rate of flavonoids from the emulsion system what finally results in better cosmetic activity of these active substances.
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The strategy to identify cadmium deactivation mechanism in Arabidopsis thaliana has been developed using selective and sensitive hyphenated techniques. Cadmium concentrations, in main parts of the plant, were determined by ICP-MS and total amount was found as 0.43–0.44 μg g−1 in leaves and 3.3–3.4 μg g−1 in roots. Speciation of the metal complexes in cells was investigated by SEC–ICP-MS in order to estimate the accumulation process. Phytochelatins, desglycyl-phytochelatins and phytochelatins homologues lacking the N-terminal γ-linked glutamic acid were extracted from plant and were identified by RPLC–ESI-MS. Two-dimensional chromatography allowed to link the metal complexes separated by SEC with isoforms of phytochelatins analyzed by high resolution RPLC and confirm their significant responsibility for metal accumulation. The potential of the cadmium complexes speciation indicates that obtained results could be reliable source of knowledge to confirm the information coming from the well-known genomic sequence of Arabidopsis and to estimate the role of γ-glutamyl transpeptidase in metabolism of glutathione.
Article
Glycyl-L-histidyl-L-lysine (GHK) is a tripeptide with affinity for copper(II) ions and was isolated from human plasma. This peptide appears to play a physiological role in wound healing. We report the stimulating effect of GHK-Cu on collagen synthesis by fibroblasts. The stimulation began between 10−12 and 10−11 M, maximized at 10−9 M, and was independent of any change in cell number. The presence of a GHK triplet in the α2(I) chain of type I collagen suggests that the tripeptide might be liberated by proteases at the site of a wound and exert in situ healing effects.
Article
In mammalian organisms copper can be found mainly in the form of complex with specific tripeptide, GHK-Cu (glycyl-l-histidyl-l-lysine-Cu(II)). GHK-Cu is the basic form in which copper is transported in tissues and permeates through cell membranes. The penetration ability of GHK-Cu through the stratum corneum and its role in copper ions transport process is the key issue for its cosmetic and pharmaceutical activity. The permeability phenomenon was studied by use in vitro model system-Flynn diffusion cell with the liposome membrane. The earlier studies on the influence of different ligands on the migration rate of copper ions through model membrane provide evidence for hampering role of ligands structure and pH of formulations in this process. Structures of copper complexes formed in solutions of different pH media were evaluated by use of ESI-MS. The permeability coefficients of copper complexes increase with increasing pH. It was proved that only tripeptide GHK and its complexes with copper: GHK-Cu and (GHK)(2)-Cu are able to migrate through membrane model of stratum corneum.
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The reactions between some Ni(II) oligopeptides (Gly-His-Lys, (Gly)4, Asp-Ala-His-Lys, Gly-Gly-His, beta Ala-His, and serum albumin) and reduced oxygen species have been characterized by spin-trapping experiments using DMPO and Me2SO. Most of the peptides possessed superoxide dismutase- and catalase-like activities leading to the formation of either oxene [NiO]2+ or, in the case of beta Ala-His, hydroxyl radicals. Both these species may affect DNA integrity through distinct mechanisms.
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Angiogenic capacity, i.e. the property of inducing neovascularization is the rule for most neoplastic cells but the exception for normal tissues. When neoplastic cells from a V2 carcinoma were implanted in the vitreous body of a rabbit’s eye and their location was more than 2 mm from the retina, a nodule developed that reached a diameter of less than 1 mm and remained practically unchanged for weeks. However, if this nodule drifted towards the retina, within a few days it was colonized by microvessels and an explosive growth followed with formation of a solid tumor that destroyed the eye. Similar effects were observed when a mouse glioma was implanted in the rabbit vitreous [1].
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Because glycosylation-dependent melanization inhibition induced in cultured B-16 melanoma cells by glucosamine is reversible, producing synchronized initiation of melanogenesis after its removal, we have analyzed the possible dynamics of initial melanogenesis through their interruption by glutathione. The addition of glutathione at 0.2% concentration to the theophylline-stimulated recovery process completely interrupts the initiation of melanization for at least 72 h. At the electron microscopic level, theophylline-treated cells have many vacuolar melanosomes with distinct pigmentation which contain some vesicles (64% of total premelanosomes) or amorphous, filamentous, or granular materials within the interior which are suggestive of pheomelanotic melanosomes. The addition of glutathione induces a complete absence of melanization in the premelanosomes, within which a filamentous interior with periodicity is generally re-formed with almost complete disappearance of internal vesicles, providing dramatic changes to the size and shape characteristic of eumelanotic melanosome. Electron microscopic dopa reaction of glutathione-treated cells shows a predominant localization of tyrosinase activity in the Golgi-associated endoplasmic reticulum-lysosome and coated vesicles, but not in premelanosomes, in contrast to their dispersed distribution in all melanogenic organelles in the theophylline-treated control, suggesting a lack of tyrosinase translocation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of tyrosinase in the large granule fraction shows that in analogy with electron microscopic observations, glutathione blocks the reappearance of membrane-bound T3 tyrosinase which occurs in the theophylline-treated control during the recovery process, whereas the dynamics of T1 tyrosinase is almost the same as that of the control. These findings suggest that glutathione provides a new situation of interrupted melanogenesis in which melanization cannot proceed despite complete formation of melanosome matrix structure and a lack of inhibition of cellular metabolisms including protein glycosylation.