Effect of oligoarginine conjugation on the antiwrinkle activity and transdermal delivery of GHK peptide

Article abstract of DOI:10.1002/psc.3234

GHK (Gly-His-Lys), a natural peptide found in human skin and plasma, has been widely used in the cosmeceutical and pharmaceutical fields. The hydrophilic GHK and GHK-Cu are limited in their abilities to penetrate deeply into skin; because of this, various strategies for their skin delivery have been developed. In this investigation, Arg₄ was conjugated with GHK to get heptapeptide, GHK-R4, and then in vitro antiwrinkle activity and transdermal delivery were compared between GHK and GHK-R4. Notably, Arg₄ conjugation accelerated the cellular penetration of GHK both in vitro and in vivo. Furthermore, higher in vitro antiwrinkle activity and collagen biosynthesis was obtained with GHK-R4 at much lower doses than with control R4-free GHK. The enhanced activity and delivery of GHK-R4 might be due to the cell penetrating ability and matrix metalloproteinase (MMP) inhibitory activity of R4 itself.

Full text of DOI:10.1002/psc.3234

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Received: 24 February 2019
DOI: 10.1002/psc.3234
Revised: 6 October 2019
Accepted: 4 November 2019
R E S E A R C H A R T I C L E
Effect of oligoarginine conjugation on the antiwrinkle activity
and transdermal delivery of GHK peptide
Ga‐Hee Hur1
Sang‐Cheol Han2 A‐Reum Ryu1
Youngsic Eom1 Jhin‐Wook Kim2
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Mi‐Young Lee1,3
1 Department of Medical Sciences,
Soonchunhyang University, Asan, Republic of
Korea
GHK (Gly‐His‐Lys), a natural peptide found in human skin and plasma, has been
widely used in the cosmeceutical and pharmaceutical fields. The hydrophilic GHK
and GHK‐Cu are limited in their abilities to penetrate deeply into skin; because of
this, various strategies for their skin delivery have been developed. In this investiga-
tion, Arg4 was conjugated with GHK to get heptapeptide, GHK‐R4, and then in vitro
antiwrinkle activity and transdermal delivery were compared between GHK and
GHK‐R4. Notably, Arg4 conjugation accelerated the cellular penetration of GHK both
in vitro and in vivo. Furthermore, higher in vitro antiwrinkle activity and collagen bio-
synthesis was obtained with GHK‐R4 at much lower doses than with control R4‐free
GHK. The enhanced activity and delivery of GHK‐R4 might be due to the cell pene-
trating ability and matrix metalloproteinase (MMP) inhibitory activity of R4 itself.
2 Department of Material Laboratory,
Dermafirm Co., Ltd., Wonju, Republic of Korea
3 Department of Medical Biotechnology,
Soonchunhyang University, Asan, Republic of
Korea
Correspondence
Mi‐Young Lee, Department of Medical
Biotechnology, Soonchunhyang University,
Asan, Chungnam 31538, Republic of Korea.
Email: miyoung@sch.ac.kr
Funding information
Soonchunhyang University; National Research
Foundation of Korea, Grant/Award Number:
2018H1D2A2010318
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1
INTRODUCTION
passively permeate through the skin.2 Thus, hydrophilic GHK and
GHK‐Cu have difficulty in penetrating into skin,7 although they can
GHK (Gly‐His‐Lys), a naturally occurring peptide motif in human skin,
plasma, and urine, has been used for wound healing and skin care.1
The GHK sequence is not only present in the alpha 2 (I) chain of type
I collagen but also released from damaged proteins by proteolysis
during wound healing.2 GHK has a strong affinity for copper, forming
GHK‐Cu, improving its bioavailability. Copper plays a prime role in the
stimulation of several enzymes associated with tissue repair and col-
lagen biosynthesis.3,4 The biological activities of GHK and copper‐
bound GHK (GHK‐Cu) include skin regeneration, wound healing, anti-
oxidant, anti‐inflammation, antilung injury, DNA/protein repair, and
anticancer.1,5
pass through the lipid barrier of the stratum corneum and reach epi-
dermal cells.8
In both cosmetic and drug delivery, extensive work has been done
to overcome the barrier for dermal or transdermal delivery, facilitating
more efficient penetration into the deeper layers of the skin, sufficient
to reach the systemic circulation.9-11 While there are many reports on
GHK‐Cu to date, fewer data on GHK without copper are available.
However, all the available evidence is consistent with GHK exerting
its biological effects as GHK‐Cu.2
Currently, incidences of contact dermatitis by copper exposure
have been reported, although GHK‐Cu has a low potential for skin
irritation.12 Considering the diverse cosmeceutical potential of
GHK, it is important to provide safest and most efficient alternative
with enhanced skin absorption and biological activity. Therefore, we
investigated whether Arg4 conjugation might accelerate the skin
penetration of GHK in vitro and in vivo. Furthermore, the enhance-
ment of antiwrinkle activity and collagen biosynthesis of GHK
via Arg4 conjugation in UVB‐induced fibroblasts were also
investigated.
A wide variety of data show that GHK is able to bind not only
with copper but also with heparin and heparin sulfate, facilitating its
involvement in cell adhesion, extracellular matrix structure, the stimu-
lation of cell migration, and differentiation.6 GHK (with or without
copper) is functional starting from picomolar concentrations; how-
ever, much higher levels of GHK have been required clinically
because of its low delivery through skin.2 As a general rule, only lipo-
philic molecules with a molecular weight below 500 Da are able to
J Pep Sci. 2019;e3234.
wileyonlinelibrary.com/journal/psc
© 2019 European Peptide Society and John Wiley & Sons, Ltd.
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https://doi.org/10.1002/psc.3234

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HUR ET AL.
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treatment for 24 hours, Hs68 cells were treated with MTT [3‐(4,5‐
dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] to determine
viability. After formazan formation by MTT, 50 μL DMSO was added
and the absorbance was measured at 570 nm. The proliferative effects
of the peptide on Hs68 cells were determined by WST assay. Briefly,
cells were cultured for 24 hours, and then cells in DMEM containing
1% FBS were treated with various concentrations of peptides for 48
or 72 hours. WST‐1 solution diluted in phosphate‐buffered saline
(PBS) at a ratio of 1:10 was then reacted with the cells in the dark
for 3 hours. The absorbance was measured at 450 nm.
2
MATERIALS AND METHODS
Peptide synthesis
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2.1
All peptides were synthesized using standard Fmoc solid‐phase peptide
synthesis with 2‐chlorotrityl (CTL) resin. The procedure includes three
steps: synthesis of H‐Arg (Pbf)Arg (Pbf)Arg (Pbf)Arg (Pbf)‐CTL‐resin 1,
synthesis of H‐ArgArgArgArg‐OH 2, and synthesis of H‐GlyHisLys‐
ArgArgArgArg‐OH 3. To synthesize H‐Arg (Pbf)Arg (Pbf)Arg (Pbf)Arg
(Pbf)‐CTL‐resin 1, 7 g (10 mmoles) CTL resin (BeadTech, Korea, 1%
DVB cross‐linked, 100‐200 mesh, 1.41 mmol/g) was added to the reac-
tor and then swelled using methylene chloride (MC) and
dimethylformamide (DMF) sequentially. After reaction with Fmoc‐Arg
(Pbf)‐OH, 6.4 g (10 mmol, 1 eq) and N,N′‐diisopropyl ethylamine
(DIPEA) 5.3 mL (3 eq) in DMF 40 mL. After the capping reaction in
MC: MeOH: DIPEA (17:2:1), deblocking was performed in 20% piperi-
dine in DMF. The elongation of Arg (Pbf) was performed using HBTU (3‐
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2.3
Fluorescent labeling of peptide with FAM
FAM (6‐carboxyfluorescein) in DMSO was slowly added dropwise into
the peptide solution in 0.1 M bicarbonate buffer pH 9.0. The molar
ratio of FAM to peptide was 1:5. The reaction mixture was incubated
at room temperature (RT) for 2 hours in the dark and then dialyzed
and concentrated.
[bis
(dimethylamino)methyliumyl]‐3H‐benzotriazol‐1‐oxide
hexafluorophosphate) and HOBt (N‐hydroxybenzotriazole). To synthe-
size H‐ArgArgArgArg‐OH (R4) 2, cleavage and deprotection of 1 was
performed in a solution of 95% trifluoroacetic acid (TFA), 2.5% MC,
and 2.5% water for 3 hours. After the resin was filtered and washed with
a small volume of MC to collect the cleaved peptide in filtrate, the com-
bined filtrate was then evaporated under reduced pressure, and the
resulting residue was precipitated in 100 mL of cold ether. After filter-
ing, the crude peptide was dissolved in 40 mL distilled water and puri-
fied by Prep‐LC (column ID 5 cm) and then freeze dried to yield 2 (1.7
g, yield 26.6%, purity 99.1% by high‐performance liquid chromatogra-
phy [HPLC]) as a white powder. The molecular weight of the final prod-
uct was measured as 643.0 (M+1, calculated MW: 642.4) by liquid
chromatography–mass spectrometry (LC‐MS). To synthesize H‐
GlyHisLys‐ArgArgArgArg‐OH 3, the peptide on resin 1 was elongated
further with protected amino acids (Fmoc‐Lys (Boc)‐OH, Fmoc‐His
(Trt)‐OH, and Boc‐Gly‐OH in that order. Resin 3 was cleaved and
deprotected in cleavage cocktail solution for 3 hours; the crude peptide
was purified with Prep‐LC (column ID 5 cm) and then freeze dried to
yield 4 (3.6 g, yield 37.3%, purity 99.2% by HPLC) as white powder.
The molecular weight of the final product was measured as 965.60 (M
+1, calculated MW: 964.59) by LC‐MS. The synthesized peptides were
acetate form of GHK (GHK‐acetate), GHK‐R4, and R4.
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2.4
In vitro penetration of GHK and GHK‐R4
The cellular uptake of GHK and GHK‐R4 in Hs68 fibroblast was com-
pared upon conjugating with FAM. Hs68 cells were treated with 50
μM fluorescent GHK and GHK‐R4 for 1 or 3 hours. The cells were
washed twice with PBS and then filled with DMEM until observed.13
The prepared cells were then observed by confocal microscope (Olym-
pus, Tokyo, Japan), and the images were recorded.
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2.5
In vivo penetration of GHK and GHK‐R4
The protocol was approved by the Animal Research Ethics Committee
at Soonchunhyang University, approval number: SCH18‐0062. SKH‐1
hairless mice (male, 6 weeks old) were obtained from Orient Bio
(Seongnam, Korea). On the day of the experiment, GHK and GHK‐
R4 in DMEM were applied to approximately 2 cm2 of the skin on
the back of each mouse. At 1, 2, 4, and 6 hours postapplication, the
mice were euthanized and the area of application was dissected. The
sample was placed in 10% formalin for 24 hours, then embedded in
Tissue‐Tek OCT compound (Sakura Finetek, Torrance, California),
and sectioned using a cryostat microtome (Leica, Wetzlar, Germany).
The skin sections (14 μm) were mounted on glass slides. The slides
were visualized without any additional staining or treatment with a
10× objective using a confocal microscope (Olympus, Tokyo, Japan)
equipped with a filter for Alexa Fluor488 and FV10‐ASW software.
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2.2
Cell culture, viability, and proliferation
Hs68 human dermal fibroblasts were purchased from the American
Type Culture Collection (Manassase, Virginia) and cultured in mono-
layers at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum (FBS). Hs68 cells (1 ×
105 cells/well) were seeded in a 6‐well plate (Falcon, Corning, New
York) for 24 hours and then UVB irradiated (200 mJ/cm2, 312 nm)
(VL.215‐LM, Vilber Lourmat, Eberhardzell, Germany). After UVB irradi-
ation, the cells were treated with various concentrations of peptides in
medium containing 1% serum for 48 hours. After GHK and GHK‐R4
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2.6
Sircol collagen assay
Total soluble collagen in Hs68 cell culture supernatant was quantified
using the Sircol collagen assay (Biocolor, Belfast, UK). UVB‐irradiated
Hs68 cells were incubated for 48 hours with GHK or GHK‐R4. One
milliliter of Sirius red dye, an anionic dye that reacts specifically with
the basic side chain groups of collagens under assay conditions, was

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HUR ET AL.
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added to 400 μL of cell culture medium supernatant and incubated
with gentle rotation for 30 minutes at RT. After centrifugation, the
pellet was washed with ice‐cold acid‐salt wash reagent, released in
alkali reagent, and the absorbance at 570 nm was measured by ELISA
(Sunrise, Tecan, Männedorf, Switzerland). The amount of collagen was
calculated on the basis of the standard curve obtained with bovine
type I collagen supplied with the kit.
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2.7
Elastase inhibition assay
Elastase inhibition was measured using porcine pancreatic elastase
(Sigma, St. Louis, Missouri) and N‐succinyl‐Ala‐Ala‐Ala‐p‐nitroanilide
as a substrate. The reaction mixture contained 200 mM Tris‐HCl
buffer (pH 8.0), 0.1 U/mL elastase, and 3.2 mM N‐succinyl‐Ala‐Ala‐
Ala‐p‐nitroanilide. The mixture was incubated for 10 minutes at 37°
FIGURE 1 The scheme for the synthesis of
GHK (GHK‐acetate form) and GHK‐R4 by
using standard Fmoc chemistry‐based solid
peptide synthesis with 2‐chlorotrityl resin
FIGURE 2 (A) Effect of cell viability of GHK and GHK‐R4 on Hs68 fibroblast cells. Cell viability was determined by MTT assay. ***P < .05 compared
with control. (B) Proliferative activity of GHK and GHK‐R4 on the proliferation of fibroblasts, measured by the WST‐1 assay. *P < .05 compared with
control

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HUR ET AL.
C after adding the peptide. The release of p‐nitroaniline was measured
at 410 nm using a 96‐well plate reader.
2.5% Triton X‐100 on a shaker for 1 hour at RT to remove the SDS.
The gel was then incubated in 50 mL reaction buffer (50 mM Tris‐
HCl, pH 7.5, containing 10 mM CaCl2 and 0.15 M NaCl) at 37°C over-
night, stained with brilliant blue R‐250, and destained with methanol‐
acetic acid in distilled water.
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2.8
Gelatin zymography
MMP‐2 secreted into the culture medium was measured by gelatin
zymography. Hs68 fibroblast cell medium was collected after GHK,
GHK‐R4, and R4 treatment. The supernatant samples were mixed with
standard SDS‐PAGE gel loading buffer without β‐mercaptoethanol
and loaded on the gel without heat denaturation. Samples were sepa-
rated by SDS‐PAGE through 8% gel containing 0.1% gelatin at 90 V
for 2 hours. After electrophoresis, the gel was washed twice with
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2.9
Western blot analysis
Whole‐cell lysates were prepared, and samples containing 25 μg of
total protein were separated on 8% to 10% SDS‐PAGE gels and then
transferred onto polyvinylidene difluoride (PVDF) membranes for 1
hour at 400 mA. After transfer, the membranes were blocked for 2
FIGURE 3 (A) Effect of GHK and GHK‐R4 on the MMP‐2 activity measured by gelatin zymography, (B) on the mRNA level of MMP‐2 and MMP‐
9 by RT‐PCR, and (C) the protein expression level of MMP‐2 and MMP‐9 by western blot. Results are expressed as mean SD of independent
*
three tests. P < .05 compared with UVB‐irradiated control