RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Evaluation of a Highly Skin Permeable Low-Molecular-Weight

Protamine Conjugated Epidermal Growth Factor for Novel Burn
Wound Healing Therapy
JI HAE LEE,1 IL-HONG BAE,1 JIN KYU CHOI,2 JIN WOO PARK3,4
1
Amorepacific Corporation R&D Center, Giheung-gu, Yongin-si, Gyeonggi-do, 446-729, Republic of Korea
2
R&D team, Pacificpharma Company, Anseong-si, Gyeonggi-do, 456-370, Republic of Korea
3
College of Pharmacy, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam, 534-729, Republic of Korea
4
Natural Medicine Research Institute, College of Pharmacy, Mokpo National University, 1666 Youngsan-ro, Muan-gun, Jeonnam,
534-729, Republic of Korea

Received 27 June 2013; revised 13 August 2013; accepted 13 August 2013

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23725

ABSTRACT: We evaluated the laser induced burn wound healing efficacy of a recombinant low-molecular-weight protamine conjugated
epidermal growth factor (rLMWP–EGF). rLMWP–EGF was prepared by genetically combining LMWP with the N-terminal sequence of EGF;
we obtained a homogeneous modified EGF without reduced biological activity. Because of the protein transduction domain of LMWP,
rLMWP–EGF showed enhanced drug penetration across artificial skin constructs and excised mouse skin layers versus EGF and showed
significantly improved burn wound healing efficacy, with accelerated wound closure and minimized eschar and scar formation, compared
with EGF or no treatment. Histological examination also revealed that rLMWP–EGF permeated through the intact skin around the wound
and facilitated residual epithelial cell proliferation in an integrated manner to reform an intact epidermis. Radiofrequency microwound
formation was effective for reducing large hypertrophic scars formed after severe laser burning by collagen remodeling but rLMWP–EGF
did not show a meaningful synergistic effect in burn scar reduction. However, rLMWP–EGF was helpful for forming skin with a more
normal appearance and texture. Thus, rLMWP–EGF demonstrated therapeutic potential as a novel topical burn wound healing drug with
no obvious toxic effect. 
C 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci

Keywords: epithelial delivery/permeability; wound healing; percutaneous; protein delivery; peptide delivery; transdermal drug delivery;
conjugation; permeability; permeation enhancers

INTRODUCTION stimulate cell proliferation and synthesis of new extracellular

matrix (ECM).6–8
The healing of wounds is one of the most complex and interac-
To date, various growth factors have been examined for their
tive biological processes, involving inflammation, proliferation,
potential as wound therapeutics, such as transforming growth
angiogenesis, reepithelialization, tissue regeneration, and re-
factor-$ and vascular endothelial growth factor,9–11 which accel-
modeling. In particular, burns are special types of wounds that
erate the healing of wounds in animal models. Platelet-derived
require specialized management protocols and are classified ac-
growth factor has also been shown to accelerate the healing of
cording to the depth of injury. Burns to the skin or other tissues
human diabetic foot ulcers, and basic fibroblast growth factor
produce a markedly different healing response because of their
has been approved as a treatment for wounds in Japan.12,13 Sev-
effects on the viability of cells and tissues.1,2 Thermal burns,
eral other growth factors, including granulocyte-macrophage
in particular, create an extensive zone of frank necrosis that
colony-stimulating factor and epidermal growth factor (EGF),
includes dead cells and denatured or even charred connective
have shown benefits in clinical trials.14,15 Some reports regard-
tissue. Beyond the area of total destruction, a zone of coag-
ing growth factors in burn wound healing have suggested that
ulation necrosis exists, in which denaturation of plasma and
they may play an important role in the healing process.16–18
cellular proteins leads to the obstruction of blood vessels and
Of the various growth factors described above, EGF is a
lymphatics. This effect, in turn, induces nutrient starvation in
polypeptide composed of 53 amino acids that accelerates epi-
the involved tissue.3
dermal and mesenchymal regeneration, cell motility, and cel-
Numerous cells release pro-inflammatory and anti-
lular proliferation. EGF acts by binding to the EGF receptor, a
inflammatory cytokines, and various growth factors modu-
tyrosine kinase, thereby initiating a series of events that regu-
late the wound healing process and accelerate or slow tissue
lates cell proliferation.19,20 The role of EGF in wound healing in
repair.4,5 Among them, growth factors act as critical extracel-
human and animals has been investigated extensively. In addi-
lular signals that orchestrate the wound healing process by
tion, topical administration of EGF accelerates dermal regen-
binding to specific high affinity receptors on the cell surface to
eration of partial-thickness or second-degree burn wounds.21–24
Currently, at least three companies worldwide are marketing
Correspondence to: Jin Woo Park (Telephone +82-61-450-2704; Fax: +82-61- EGF-based formulations to treat burns.
450-2689; E-mail: jwpark@mokpo.ac.kr)
Ji Hae Lee and Il-Hong Bae equally contributed as first authors to this work. However, most products are applied topically to the wound;
Journal of Pharmaceutical Sciences thus, the efficacy of EGF products is critically limited by its poor

C 2013 Wiley Periodicals, Inc. and the American Pharmacists Association transdermal permeability and biological stability. Many studies

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 1

2 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
have suggested that the local concentration of EGF needs to be
sufficiently high and prolonged for effective wound healing.25–27
Thus, many delivery systems have been developed to obtain
high concentrations and extended release of EGF, including
hydrogels, sponges, polymeric pellets, eletrospun nanofibers,
microspheres, a coacervate delivery system with heparin, and
a biomimetic delivery system that incorporates EGF with bio-
compatible ECM components such as hyaluronic acid, collagen,
and vitronectin.28–34 However, these systems still have limita-
tions, such as poor loading efficacy and penetration through
the skin, and reductions in bioactivity during preparation and
after application.35–37
Here, we introduce a powerful percutaneous delivery system
that enhances skin penetration, which is otherwise a strong
barrier to drug efficacy, using a genetically designed conjuga-
tion of a protein transduction domain (PTD) to EGF. We can
fully preserve EGF’s original biological activity during prepa-
ration and facilitate rapid and highly localized delivery of EGF
around the wound area. In a recent study, we genetically con-
jugated a highly positively charged low-molecular-weight pro-
tamine (LMWP), a nontoxic, arginine-rich cell translocating se-
quence (VSRRRRRRGGRRRR) in protamine, to the N-terminal
of EGF (rLMWP–EGF), and demonstrated its markedly im-
proved skin permeability in vitro. In addition, topically applied
rLMWP–EGF was significantly effective for accelerating wound
healing in full thickness and diabetic wound models.38
In the present study, we prepared a poloxamer gel formula-
tion of rLMWP–EGF and demonstrated its enhanced skin pen-
etration and efficacy for the treatment of laser-induced burn
wounds in mice. We also evaluated the synergistic effect of
radiofrequency (RF)-induced microwound formation with top-
ical administration of rLMWP–EGF in reestablishing post-
burn scarring by percutaneous collagen induction. Finally, we
conducted dermal irritation experiments in rabbits and skin
sensitization tests in mice using rLMWP–EGF in compliance
with Organization for Economic Cooperation and Development
(OECD) guidelines to evaluate any toxicological effects because
of its increased skin penetration.
MATERIALS AND METHODS
Materials
Recombinant human EGF (rEGF) and LMWP were supplied
by BIO FD&C (Whasun, Jeonnam, South Korea). A polyethy-
lene polyoxypropylene block copolymer (poloxamer 188) was
obtained from BASF for the topical gel formulation (Lud-
wigshafen, Germany).
Animal Care
The animals were acclimatized for 1 week in an animal fa-
cility under controlled conditions of temperature (23 ± 3◦ C),
relative humidity (55 ± 10%), and light (12/12 h light/dark,
with no ultraviolet exposure). The animals had free access to
a laboratory diet (Purina Company, St. Louis, Missouri) and
ion-sterilized tap water. All experiments were performed in ac-
cordance with the Pacific Pharma Institutional Animal Care
and Use Committee and the OECD “Guide for the Care and
Use of Laboratory Animals.”
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES
Methods
Preparation of rLMWP–EGF.
rEGF carrying LMWP at its N-terminus was prepared with
an expression plasmid that would overproduce a protein con-
taining LMWP–EGF in Escherichia coli, as described previ-
ously. Briefly, cDNA for LMWP–EGF was constructed by se-
rial polymerase chain reaction (PCR) of the successive addition
of amino acids of LMWP to the EGF gene (GenBank Acces-
sion No. P01133) as the first template. Then, the LMWP–EGF
gene fragment was amplified by PCR to clone the cDNA for
LMWP–EGF between NdeI and XhoI restriction sites. The for-
ward primer was 5 -CATATGGTGAGCCGTAGACGTAGACG-
3 and the reverse primer was 5 -CTCGAGTCATTAGCGCAG-
TTCCCACCACTTCAGGTCTCGGT-3 . The resulting PCR
product was sub-cloned into the TOPO vector. After plasmid pu-
rification and double digestion with NdeI/XhoI, the insert DNA
fragment was cloned into a pET-41b(+) expression vector. Then,
the correct expression vector, pET-41b(+)-LMWP–EGF, was
transformed into E. coli competent cells (BL21-DE3). BL21-
DE3 cells harboring pET-41b(+)-LMWP–EGF were grown in
2.0 L LB medium supplemented with 100 :g/mL ampicillin at
37◦ C until the optical density of the culture at 600 nm was
0.6–0.8. Protein expression was induced by adding isopropyl-
$-D-thiogalactopyranoside (1 M, IPTG) and the culture was in-
cubated overnight at 100 rpm and 25◦ C. After cell lysis, the
supernatant containing rLMWP–EGF was purified by applica-
tion to an ion-exchange column packed with Q-cellulose and a
Sephadex G-100 column.
The purity of rLMWP–EGF was analyzed using sodium do-
decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
with Coomassie blue staining and the protein concentration
was determined using the Micro BCA Protein Assay kit with
bovine serum albumin as the standard (Pierce, Rockford, Illi-
nois). The N-terminal amino-acid sequence of the purified
rLMWP–EGF was analyzed using an Applied Biosystems Pro-
cise 491 protein sequencer (Courtaboeuf, France) to confirm the
LMWP conjugation.
Biological Activity of rLMWP–EGF.
In vitro biological activity of rLMWP–EGF was evaluated us-
ing a cell proliferation assay in a mouse fibroblast cell line
(NIH3T3) seeded at a density of 5 × 103 cells per well in
100 :L Dulbecco’s modified Eagle’s medium (DMEM; Lonza,
Zurich, Switzerland) containing 10% fetal bovine serum (FBS;
Gibco, Grand Island, New York) and 1% penicillin/streptomycin
(Gibco). Then, the culture was incubated at 37◦ C for 24 h. The
serum concentration in the medium was lowered to 0.05%, and
the cells were incubated for a further 24 h. The cells were
stimulated with 1, 10, 100, and 1000 nM rEGF or rLMWP–
EGF in low-serum medium (0.5% FBS) for 24 h. To determine
cell proliferation activity, the WST-1 assay (Roche Diagnostics,
Mannheim, Germany) was performed according to the instruc-
tion manual. Diluted WST-1 solution [5 mg/mL in phosphate-
buffered saline (PBS)] was added and incubated for 2 h. The ab-
sorbance was measured with a spectrophotometer (Molecular
Devices, Sunnyvale, California). Cell viability was calculated
compared with the control.
DOI 10.1002/jps.23725
 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
In Vitro Permeabilities Through Artificial Skin.
To evaluate the penetration enhancement efficacy of EGF fol-
lowing genetic conjugation with LMWP, 30 :L rEGF, a physical
mixture of LMWP with rEGF, and rLMWP–EGF were added to
the stratum corneum (SC) side of a reconstituted human epi-
dermis model (Keraskin, MCTT Company, Seoul, South Korea)
with 0.67 cm2 in the permeation area at concentrations equiva-
lent to 12 and 60 :g/mL of rEGF, which showed no cytotoxicity
(cell viability > 90%), and then were incubated at 37◦ C in 5%
CO2 . After 24 h, the rEGF concentration was quantified using
a solid-phase sandwich enzyme-linked immunosorbent assay
(ELISA) at 450 nm (Invitrogen, Carlsbad, California). In the
case of rLMWP–EGF, 20 :L aliquots from each sample were
injected into an HPLC system equipped with a Gemini 10 :m
C18 column (250 × 4.6 mm; Phenomenex Inc., Torrance, Cal-
ifornia) and the samples were chromatographed using an iso-
cratic mobile phase of acetic acid (1→1000)-acetonitrile (20:80)
at a flow rate of 0.8 :L/min. The quantification of rLMWP–EGF
was carried out at 210 nm.
In Vitro Permeabilities Through Skin PAMPA.
In vitro artificial skin permeabilities of rEGF and rLMWP–
EGF were also evaluated using the Skin PAMPA Explorer Test
System (PN 120663; Pion Inc.). Each well of the top (accep-
tor) compartment of a STIRWELL PAMPA sandwich plate had
been pre-coated with 10 :L skin-mimetic artificial membrane
components (certramide, cholesterol, stearic acid, and silicone
oil) dissolved in chloroform. The lipid mixture solvent was then
evaporated completely. For the assay, the top (acceptor) plate
was hydrated with 200 :L diluted hydration solution (pH 7.4)
overnight. Before forming the sandwich, the bottom (donor)
plate was prefilled with 200 :L rEGF or rLMWP–EGF dis-
solved in the Prisma-HT buffer solutions (pH 7.4) at a con-
centration of 100 :g/mL. The acceptor plate was also filled
with 200 :L Prisma-HT buffer solution at pH 7.4, and then
the resulting sandwich was incubated at room temperature.
After 4, 6, 10, and 24 h, each PAMPA sandwich was sepa-
rated and 150 :L of both the donor and acceptor was collected.
The rEGF concentration was quantified using a solid-phase
sandwich ELISA at 450 nm and the permeated concentration
of rLMWP–EGF was determined using the HPLC system at
210 nm, as described above.
In Vitro Permeabilities Through Hairless Mouse Skin.
The permeabilities of rEGF and rLMWP–EGF through excised
hairless mouse skin (Han-Lim Experimental Animal Research
Center, Hwaseong, South Korea) were measured using a Franz
diffusion cell system (Laboratory Glass Apparatus, Fine Sci-
ence, Seoul, South Korea). The diffusion area was 0.785 cm2 .
The receptor phase compartment was filled with 5.5 mL PBS
(pH 7.4) and skin samples were mounted on the receptor phase
compartments with the SC facing upward. Then, the donor
compartments were clamped in place. Before adding the test
formulations to the donor compartment, the cells were allowed
to equilibrate for at least 30 min and the integrity of the epi-
dermal membranes was inspected visually. After equilibrium,
200 :L test solution made at 30 :g/mL in poloxamer 188 (6%,
w/v) was added to the donor compartment and the receptor com-
partment was stirred at room temperature with a water circu-
lating system to maintain a skin surface temperature of 32◦ C
throughout the experiment. Then, 200 :L of the receptor phase
DOI 10.1002/jps.23725
3
was withdrawn after 1, 3, 6, 9, 12, and 24 h and each aliquot
was replaced with fresh PBS. The withdrawn samples were
filtered through a membrane filter (0.45 :m, polyvinylidene
difluoride) and kept at 4◦ C until analysis. Then, the samples
from the skin permeation experiments were quantified using a
solid-phase sandwich ELISA (Invitrogen) at 450 nm for rEGF
and the rLMWP–EGF was quantified using the HPLC system
at 210 nm, as described above.
In Vivo Laser Induced Burn Wound Healing Study.
To evaluate the laser induced burn wound healing efficacy of
rEGF–LMWP, 7-week-old female hairless mice (Orient Com-
pany, Ltd., Gyunggi-do, South Korea) were anesthetized with
isoflurane (4%) and the dorsal skin was sterilely prepared with
betadine. Then an ablative fractional CO2 laser (eCO2; Lutronic
Corporation, Ilsan, South Korea) was used to irradiate the dor-
sal skin seven times. The output fluence of the CO2 laser was
40 mJ/cm2 and the density of ablation canals was 300/cm2 . The
diameter of each fractional laser was 120 :m and the overall
burn wound size was about 8 mm in diameter. After laser treat-
ment, the back of each mouse was injured, then sterilized with a
povidone/iodine solution. After 1 day, 30 :L vehicle [poloxamer
188, 6% (w/v)], rEGF in vehicle (10 :g/mL), or rLMWP–EGF
in vehicle (10 :g/mL) was applied topically to each wound area
twice per day for 12 days. The degree of wound healing was
evaluated by measuring the wound area using a digital cam-
era (PowerShot G9; Canon, Tokyo, Japan) and an image anal-
ysis program (CellSens; Olympus, Japan). The rate of wound
healing was expressed as the percentage of remaining wound
area compared with the initial wound size. On days 3 and 12,
five animals in each group were sacrificed and the wound and
surrounding tissues were collected for histological evaluation.
The excised tissues were fixed immediately in 10% neutral
phosphate-buffered formalin and then embedded in paraffin
wax. The tissue samples were sectioned at 5 :m and stained
with hematoxylin and eosin (H&E) for standard evaluation.
In addition, the degree of reepithelialization from the wound
edge was compared under a light microscope (Bx-41; Olym-
pus). To examine activated keratinocytes in the wound mar-
gin, replicate sections were immunohistochemically stained for
cytokeratin 6 (K6) using polyclonal rabbit anti-cytokeratin 6
antibody (PRB-169P; Covance Inc., USA) as the primary an-
tibody and antirabbit horseradish peroxidase-conjugated anti-
body (ab6802; Abcam, USA) as the secondary antibody.
In Vivo Skin Rejuvenation Efficacy of rEGF–LMWP in Laser
Induced Burn Scars.
The dorsal skin of 40 female hairless mice was irradiated using
an ablative fractional CO2 laser with 100 mJ/cm2 (16 × 16 mm2
in area) under isoflurane anesthesia. Then, the mice were di-
vided into four groups (n = 10 each): vehicle, RF micropora-
tion + vehicle, RF microporation + rEGF, and RF micropo-
ration + rLMWP–EGF. Fourteen days after laser irradiation,
burn scars had formed on the backs of the mice and were heav-
ily pigmented a deep red color. From day 15, 30 :L vehicle
[poloxamer 188, 6% (w/v)], rEGF (10 :g/mL), or rLMWP–EGF
(10 :g/mL) was applied topically to the hyperpigmented scar
area once daily for 22 days.
In this experiment, we also used RF-derived microporation
treatment to create microwounds in the dermis. The RF mi-
croporator, which consists of a reusable electronic controller
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES
4 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
similar in size to an electric shaver and 64 disposal stainless
microneedle electrode arrays 1 mm in length, 250 :m in di-
ameter, and with a sharpened edge, was designed to create
50 :m diameter pores at a depth of 100 :m. Each animal with
a fractional laser derived burn scar was treated with the RF
microporator five times on the outer surface of the burn scar
(300 V/500 kHz/38 W/2 ms per treatment) before topical appli-
cation of vehicle, rEGF, or rLMWP–EGF in the RF treatment
group twice a week for 14 days.
The degree of scar reduction was evaluated by measuring the
area of the scar using a digital camera (PowerShot G9; Canon)
and an image analysis program (CellSens; Olympus). The rate
of scar reduction was assessed by the percentage of remaining
scar area compared with the initial size and the difference in
skin color between normal and scarred skin. Transepidermal
water loss (TEWL) was also measured to evaluate the integrity
of the skin barrier using a vapormeter SW4102 (Delfin Tech-
nologies, Kuopio, Finland).
Skin biopsies were taken using a biopsy punch immediately
after the in vivo evaluation and then were fixed in 10% buffered
formaldehyde solution. The samples were embedded in paraffin
wax, cut to a thickness of 6 :m, and stained with H&E.
In Vivo Dermal Irritation Study
A dermal irritation study was performed according to OECD
Guideline 404 “Acute Dermal Irritation/Corrosion (2002).” New
Zealand White male rabbits with healthy, intact skin were used
(17 weeks of age, 2.7–3.6 kg in body weight). Approximately 24 h
before the test, the hair coat was removed by closely clipping
the dorsal area of the trunk of the animals. rEGF or rLMWP–
EGF solution (100 :g/mL) was applied to the skin and covered
with a gauze patch for 24 h. At 24, 48, and 72 h after removal of
the patches, the dermal response was determined in accordance
with the guideline. The mean scores at 24, 48, and 72 h were
summed and averaged to obtain the primary irritation index
(PII).
In Vivo Skin Sensitization Study
Local lymph node assays were carried out according to the
OECD test guideline number 429 “Skin Sensitization: Local
Lymph Node Assay (2010)”. Accordingly, 8-week-old female
BALB/c mice (Orient Bio, Seongnam, Korea) were used. A pre-
liminary experiment was conducted to determine the highest
concentration because ear swelling was assessed by measuring
the ear thickness on days 1, 2, 3, and 6 (before sacrifice). In the
main study, mice were treated by topically applying test or vehi-
cle solutions to the entire dorsal surface of each ear, once daily,
for 3 consecutive days. The positive control group received 0.1%
1-chloro-2,4-dinitrobenzene (DNBC) in the vehicle. On day 5, all
mice were dosed with 5-bromo-2 -deoxyuridine (BrdU) by intra-
venous injection into the tail vein. Approximately 24 h later, all
mice were sacrificed, and the draining auricular lymph node
was excised, weighed, individually pooled for each animal (i.e.,
two lymph nodes per animal), and collected in PBS. Follow-
ing some further processing steps, the incorporation of BrdU
into the lymph nodes was assessed by flow cytometry. For each
dose group, the stimulation index (SI) was calculated as the
ratio of the mean BrdU incorporation of the test group and the
negative control group. In addition to determining the poten-
tial lymph node proliferative response, ear thickness measure-
ments and visual local skin reactions were examined to assess
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES
the potential irritation of the test materials. When ear thick-
ness increased beyond 25% compared with the control, the test
material was considered a potential irritant. A test material
was also considered a skin sensitizer when the SI for a dose
group was above 3.
Statistics
All data are expressed as means ± standard deviation. Stu-
dent’s unpaired t-test was used to compare means between
groups. A p value of less than 0.05 was considered to indicate
statistical significance.
RESULTS
Characterization of rLMWP–EGF
To conjugate LMWP to the N-terminal of rEGF, we constructed
an expression plasmid that overproduced a protein contain-
ing LMWP–EGF in E. coli. Purified proteins from each step
were analyzed by SDS-PAGE (Fig. 1a). Extra bands were de-
tected in the eluted fractions from ion-exchange chromatogra-
phy (Fig. 1a, lanes 7 and 8). However, the contaminant protein
was completely removed by gel filtration, as shown in lanes
4 and 6, and homogeneous rLMWP–EGF protein displayed a
band with an apparent MW of about 8.2 kDa. This protein rep-
resented 98% of total protein, based on densitometric quantifi-
cation of the lane using the BandScan software. The N-terminal
amino-acid sequence of the purified rLMWP–EGF was deter-
mined with a protein sequencer and the sequences of rLMWP–
EGF and rEGF were Val–Ser–Arg–Arg–Arg–Arg–Arg–Arg–
Gly–Gly–Gly–Arg–Arg–Arg–Asn–Ser–Asp–Ser and Asn–Ser–
Asp–Ser–Glu, respectively (data not shown). The sequence
results were consistent with the theoretical sequences, thus
demonstrating that LMWP was specifically conjugated to the
N-terminus of EGF as intended.
The biological activity of rLMWP––EGF was confirmed by
evaluating rLMWP–EGF-induced migration and proliferation
of NIH3T3 cells. At 1, 10, 100, and 1000 nM rLMWP–EGF, cell
viability values were 100%, 112%, 124%, and 131% higher, re-
spectively, than those of the control group. rEGF showed 101%,
101%, 113%, and 115% increases in cell proliferation, compared
with the control group at 1, 10, 100, and 1000 nM, respectively
(Fig. 1b). rLMWP–EGF exhibited slightly improved biological
activity versus rEGF, but there was no statistically significant
difference between the groups. Thus, conjugation of LMWP
caused no loss in the original biological activity of rEGF un-
der these conditions.
In Vitro Skin Permeability Studies
To evaluate the skin penetration efficacy of rLMWP–EGF,
we performed a comparative permeability study with rEGF,
LMWP + rEGF, and rLMWP–EGF using an artificial 3D
skin epidermal constructs (Keraskin). After 24 h, the cumu-
lative permeated concentrations of rEGF, LMWP + rEGF,
and rLMWP–EGF at 12 :g/mL loading concentrations were
0.59 ± 0.50 (×10−3 ), 3.76 ± 0.59 (×10−3 ), and 125.08 ± 16.42
(×10−3 ) :g/mL, respectively. The cumulative concentrations of
rEGF, LMWP + rEGF, and rLMWP–EGF at 60 :g/mL loading
concentrations were 0.67 ± 0.24 (×10−3 ), 16.83 ± 5.00 (×10−3 ),
and 1281.09 ± 138.91 (×10−3 ) :g/mL, respectively (Fig. 2a).
The permeation of each material increased in a concentration-
dependent manner. The permeated concentrations of rEGF at
DOI 10.1002/jps.23725
 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 5

Figure 1. (a) SDS-PAGE analysis after various purification steps

of recombinant low-molecular-weight protamine conjugated epidermal
growth factor (rLMWP–EGF). Markers (M), total cell lysate after in-
duction with 1 mM IPTG (lane 1), total cell lysate after cell lysis by
sonication (lane 2), loading sample on ion-exchange chromatography
(lane 3), blank (lane 5); eluted fractions from ion-exchange chromatog-
raphy and loading sample on gel filtration chromatography (lanes 7 and
8), and purified and concentrated rLMWP–EGF after ion-exchange and
gel filtration (lanes 4 and 6). (b) Migration and proliferation of NIH3T3
cells versus the control group after incubation with 1, 10, 100, and
1000 nM rEGF or rLMWP–EGF for 24 h. Each value is the mean ±
standard deviation (n = 6). *p < 0.05 versus control.

12 and 60 :g/mL were 6.4- and 25.3-fold greater, respectively,

by physical incorporation with LMWP. However, permeation
was significantly greater with rLMWP–EGF than with rEGF
and LMWP + rEGF.
We also evaluated the penetration enhancement of rLMWP–
EGF using an artificial skin membrane, skin PAMPA
(Fig. 2b). The cumulative penetrated concentrations of
rLMWP–EGF were 4.74 ± 0.36, 6.26 ± 0.69, 9.74 ± 1.81,
and 19.12 ± 0.27 :g/mL at 4, 6, 10, and 24 h, respectively.
However, the cumulative concentrations of rEGF after 4, 6,
10, and 24 h were 0.00 ± 0.00, 0.19 ± 0.09, 0.48 ± 0.03, and
0.97 ± 0.26 :g/mL, respectively. The penetration of rLMWP–
EGF through the artificial skin membrane was much faster and
its cumulative permeated concentration after 24 h was about
19.7-fold higher than that of rEGF.
Figure 2. In vitro permeation through artificial skin constructs and
hairless mouse skin. (a) Permeated concentrations of rEGF, physical
mixture of LMWP with rEGF, and rLMWP–EGF through the artifi-
cial skin construct after applying concentrations equivalent to 12 and
60 :g/mL rEGF. (b) Time-course of cumulative permeated concentra-
tions of rEGF or rLMWP–EGF through the skin PAMPA; (c) compar-
ison of cumulative permeation of rEGF or rLMWP–EGF through the
excised hairless mouse skin after 24 h. Each value is the mean ± stan-
dard deviation (n = 6). *p < 0.05 versus rEGF.
Skin penetration of rLMWP–EGF was also examined in
the excised hairless mouse. Conjugation with LMWP sig-
nificantly enhanced skin penetration of rEGF (Fig. 2c). Af-
ter 24 h, the cumulative concentration of rLMWP–EGF was
81.68 ± 34.13 (×10−3 ) :g/mL and 742.5-fold higher than that of

DOI 10.1002/jps.23725 Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES

6 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 3. rLMWP–EGF gel formulation accelerated healing of laser-induced burn wounds. (a) Burn wound closure over time, measured as
percent of original wound area. (b) Representative photographs of wounds treated with vehicle [poloxamer 188, 6% (w/v)], rEGF (10 :g/mL), or
rLMWP–EGF (10 :g/mL). Bar indicates 10 mm. (c) Eschar size over time, measured as percent of original eschar area. Each value is the mean
± standard deviation (n = 10). *p < 0.05 versus vehicle; **p < 0.01 versus vehicle.

rEGF [0.11 ± 0.03 (×10−3 ) :g/mL]. In particular, although the
permeated amount of rEGF after 24 h was only about 0.0028%
of the loaded amount, the cumulative amount of rLMWP–EGF
was 2674-fold higher (7.4873% of the loaded amount).
Effects of rLMWP–EGF in the Laser Induced Burn Wound Model
To demonstrate the burn wound healing efficacy of rLMWP–
EGF, compared with rEGF and vehicle in vivo, burn wounds
were created using a fractional CO2 laser emitting consistent
thermal energy. As shown in Figures 3a and 3b, rEGF treat-
ment (10 :g/mL) significantly reduced the burn wound area
compared with that in the control (vehicle) group. A significant
difference was observed on day 5. In contrast, rLMWP–EGF
showed more enhanced wound-healing activity throughout the
test period and significantly accelerated closure of wounds by
day 8. At days 5 and 12, 66.0 ± 2.5% and 38.2 ± 2.4% of the
wound remained in the rLMWP–EGF group, compared with
82.2 ± 2.7% and 53.0 ± 3.5% in the rEGF treatment group
and 95.1 ± 3.2% and 47.3 ± 3.2% in the vehicle group, re-
spectively. We also measured the size of the eschar during the
healing process. Eschar formation was obvious on day 4 in all
treatment groups. At day 5, 73.9 ± 5.8%, 47.5 ± 7.9%, and
35.0 ± 4.9% of the eschars in the vehicle, rEGF, and rLMWP–
EGF treatment groups, respectively, remained, and then each
decreased markedly in size in all groups. Eschar formation in
the rLMWP–EGF treatment group was about 74% further re-
duced versus that with rEGF (Fig. 3c).
Histologically, the rLMWP–EGF gel formulation signifi-
cantly promoted reepithelialization of the wound in the early
phase (Fig. 4). On day 3, the reepithelialized area over the
wound bed in the rLMWP–EGF-treated group was the largest
of the groups, about 30% larger than that of the vehicle group.
Furthermore, rLMWP–EGF treatment appeared to lead to a
thicker and denser neoepidermis layer in the burn wound than
the other treatment groups. In contrast, the reepithelialized
area treated with rEGF was 13% larger than that of the vehicle
group. It was also found that cytokeratin 6, a marker for acti-
vated keratinocytes during wound healing that is induced by
EGF,39 was highly expressed in the suprabasal compartment
nearest to the wound edge in the rLMWP–EGF-treated group.
All of these results indicate that rLMWP–EGF treatment con-
siderably improved epidermal recovery and reepithelialization
of the burn wounds versus rEGF or the vehicle.
Effects of rLMWP–EGF in the Laser Induced Burn Wound Scar
After irradiation with a high-powered fractional CO2 laser,
wounds about 2.6 cm2 in area and 250 :m in maximal depth
formed. No sebaceous gland remained in the upper dermis

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.23725

 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 7

Figure 4. rLMWP–EGF gel formulation accelerates wound reepithelialization and activates keratinocytes around the burn wound margin.
Wound sections stained with hematoxylin and eosin (H&E) for general observation of reepithelialization and immunohistochemical staining for
keratin protein, K6 (brown) as a marker of activated keratinocytes via binding with rEGF or rLMWP–EGF. Representative light microscopy
images of sections from the wound tissues at day 3. Arrows indicate newly formed epithelial layer within the burn wound legion. Bar = 200 :m.

but most of the lower dermis and hypodermis with skin ap-
pendages, including remnants of hair follicles, were intact. On
day 14, the epidermis had almost returned to normal and the
margin between the scar and intact tissue became indistin-
guishable; however, wound contracture with poor appearance
was caused by hypertrophic scarring and keloid formation. The
burn scar was significantly diminished and normalized only af-
ter treatment with postburn RF microwound formation. At day
22, 81.6 ± 5.4%, 83.8 ± 3.8%, 80.6 ± 3.5%, and 93.7 ± 3.7%
of the scar remained in the RF microporation, RF micropo-
ration + rEGF, RF microporation + rLMWP–EGF, and vehi-
cle treatment groups, respectively. Hence, RF microporation
showed significant potency for rejuvenating scarred skin and
reestablishing a more normal appearance by replacing the scar
collagen with new collagen under the epidermis (Figs. 5a and
DOI 10.1002/jps.23725
5b). Although topical administration of rLMWP–EGF combined
with RF microporation did not reduce scar size much, skin color
was significantly normalized and the level of TEWL was in-
creased more than in the other groups (Figs. 5c and 5d).
Dermal Irritation and Skin Sensitization
Skin irritation was assessed at 24, 47, and 72 h. No dermal
response, including erythema or edema, was observed in rabbits
treated with rEGF or rLMWP–EGF. The PII was also calculated
to be zero in these groups.
Ear swelling responses 24 h after challenge are shown in
Figure 6a. rEGF at 100 and 10 :g/mL both increased ear
weight by 103% compared with the vehicle control, respectively,
but rEGF at 300 :g/mL increased ear weight significantly, by
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES
8 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 5. Effect of RF microporation with topical application rLMWP–EGF gel formulation on scar reduction in the hypertrophic burn scar
model. (a) Percentages of scar area measured over 22 days. (b) Representative images of burn scar treated with vehicle, RF microporation +
vehicle, RF microporation + rEGF, or RF microporation + rLMWP–EGF. Bar indicates 10 mm. (c) Effect of RF microporation treatment with
or without topical application of rEGF or rLMWP–EGF on the TEWL in the burn scar area on day 22. (d) Difference in skin color between
normal and burn scar area on day 22. The difference in skin color was evaluated by analyzing digital images. Each value is the mean ± standard
deviation (n = 10). *p < 0.05 versus control.

110%, and thus showed a tendency to cause skin irritation. The
three concentrations of rLMWP–EGF increased ear weights by
106%, 100%, and 100% compared with the vehicle treatment,
respectively. The cell proliferation responses in the ear 24 h
after challenge are shown in Figure 6b. The SIs of rEGF at
300, 100, and 10 :g/mL were 2.2, 2.0, and 2.5, respectively. In
contrast, the maximum SI achieved in response to 300 :g/mL
of rLMWP–EGF was 1.8. rEGF showed relatively higher skin
sensitivity than rLMWP–EGF but the SI values at the three
test concentrations were less than 3, the cut-off value for skin
sensitization activity. Thus, both rEGF and rLMWP–EGF can
be considered nonsensitizing under the conditions of this test.
DISCUSSION
Our delivery system potently enhanced the skin permeability
of EGF using PTD. We site-specifically conjugated LMWP to
the N-terminal of EGF by recombinant DNA techniques; thus,
we could control the LMWP conjugation site and also obtain a
homogeneous LMWP-conjugated EGF.
The healing of skin wounds is a complex process involving
epidermal regeneration, fibroblast proliferation, neovascular-
ization, and ECM synthesis. Some studies have shown that
exogenous application of EGF accelerates and improves the
quality of burn healing.22,40,41 However, it can only effectively
accelerate wound healing when it is applied in a sustained and
localized fashion. In addition, EGF is more important in the
initial stages of wound healing because it stimulates fibroblast
and keratinocyte proliferation within 24 h after application
and significantly increases mRNA expression levels of EGF-
responsive genes, such as epiregulin, keratin, and loricrin.42,43
Our approach to improve the wound healing efficacy of EGF is
to make it more effectively and quickly absorbed through the
intact skin around the wound lesion and to stimulate fibroblast
proliferation.

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.23725

 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 9

Figure 6. In vivo skin sensitization effects of rEGF or rLMWP–EGF. (a) Ear swelling response to 300, 100, or 10 :g/mL rEGF or rLMWP–EGF.
(b) Proliferative activity of lymph nodes after application of 300, 100, or 10 :g/mL rEGF or rLMWP–EGF. Each value is the mean ± standard
deviation (n = 10). *p < 0.05, versus control.

According to in vitro permeability studies, 0.0028% and
7.4873% of the initial loading amounts of rEGF and rLMWP–
EGF penetrated excised mouse skin after 24 h, respectively. Al-
though skin penetration of different PTDs has been reported,
the exact mechanism remains unclear because the intercellular
lipid domain of the SC differs from cell membranes not only in
lipid composition but also in water content and lipid/protein ra-
tio. In addition, the outermost layers of the skin consist of non-
viable cells, so endocytosis is unlikely to be relevant. However,
the enhanced skin penetration of arginine-rich peptide, LMWP,
conjugated to EGF may be explained by the formation of hydro-
gen bonds between arginine and lipid phosphates in the mem-
brane and the interaction between arginine and the ECM. Such
interactions may lead to disruption of the ordered lipid orienta-
tion, creating a concentration gradient through open channels,
ultimately increasing the permeation of rLMWP–EGF through
the SC.44–46
In our previous study, the permeated concentration of
rLMWP–EGF through the excised hairless mouse skin was ap-
proximately 11-fold higher than that of rEGF at 24 h after
loading with PBS.38 In this study, rLMWP–EGF was formu-
lated with 6% poloxamer 188 as a delivery vehicle; this sys-
tem showed significantly increased skin permeation, compared
with that of rEGF (742.5-fold higher). The synergistic skin per-
meation enhancement using poloxamer may have been caused
by a surfactant effect. Poloxamer 188 is a synthetic nonionic
multiblock copolymer that contains a central hydrophobic core
consisting of approximately 30 units composed of propylene ox-
ide (PO) and two flanking hydrophilic moieties, each consisting
of ethylene oxide (EO). The hydrophobic PO repeat units might
adsorb, intercalating among the hydrocarbon tails of the lipid
at the interface, leaving the flanking EO repeat units immersed
in the aqueous phase. Thus, poloxamer 188 may interact with
lipid bilayers, be incorporated into the membrane, and then sol-
ubilize lipids within the SC. Furthermore, poloxamer tends to
form micelles; these micelles might interact with rLMWP–EGF
and then form a thermodynamically stable association, which
may also prevent denaturation and stabilize rLMWP–EGF.47–49

DOI 10.1002/jps.23725 Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES

10 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Immediately after laser treatment, necrotic cutaneous le-
sions showed a consistent thickness in the deep dermis above
the hypodermis. Thus, although the absorption barrier, SC, was
completely removed, the exogenously applied growth factor still
could not be directly absorbed through the wound lesion. Nev-
ertheless, the topical formulation of rLMWP–EGF significantly
decreased the laser induced burn wound area and accelerated
epidermal regeneration. We also observed more activated ker-
atinocytes, which stained positive for K6 protein, well beyond
the wound margin in the rLMWP–EGF treatment group. Thus,
rLMWP–EGF may be highly absorbed through the intact skin
around the wound margin, where growth factors are especially
necessary to stimulate reepithelialization.
The burn wound eschar is avascular and frequently several
millimeters away from the microvasculature. For topical an-
timicrobial agents to properly prevent infection of burn wounds,
penetration enhancers or hydrants should be incorporated into
topical therapeutics and/or eschar formation itself should be
minimized.50,51 Moreover, scars also most commonly occur when
epithelialization has been delayed during the healing of deep
dermal burn wounds. In this study, rLMWP–EGF treatment
also minimized eschar and scar formation because of its en-
hanced efficacy for resurfacing the wound with new epithelium,
providing rapid restoration of epidermal integrity and barrier
function.
Epidermal regeneration and dermal repair can usually close
the wound smoothly, but wound contracture caused by hyper-
trophic scar and keloid formation may also complicate the heal-
ing process and result in poor appearance, itching, and scar
contracture, with functional impairment clinically.52 The dis-
ruption of collagen organization resulting from scarring also
causes local changes to skin coloration.53–55 Thus, it is also im-
portant to remove residual cicatricial deformities from burn
scars to improve esthetic appearance and quality of life. To
rejuvenate scarred skin and reestablish a more normal appear-
ance, the maintenance or establishment of a perfect epidermis
with natural dermal papillae, good hydration, normal color,
and normal resilience is required. Various studies using tis-
sue transfer, ablative laser resurfacing, and/or deep chemical
peels have been performed.56–58 Most treatments are based on
wound formation in the dermis to stimulate new collagen for-
mation and elastin synthesis.59,60 In this study, hypertrophic
scars exhibiting follicular plugging and marked fibroplasia with
dense fibrillar collagen distributed in the plane of the epidermis
were formed. For reepithelialization, we used RF microporation
technology. The current emitted from the RF device created mi-
crowounds in the skin by vibrating and vaporizing intracellular
water molecules with minimal pain and damage in the epider-
mis and upper dermis layer. As a result, the RF microporation
treatment alone was effective for minimizing burn scars by pro-
moting the replacement of scar collagen with normal collagen
and reducing depressed and contracted scars. However, com-
bined treatment with rLMWP–EGF further diminished water
loss as healing progressed and resulted in a more normal ap-
pearance and skin texture due to its faster and enhanced reep-
ithelialization efficacy.
CONCLUSIONS
We modified EGF by genetically conjugating a PTD, LMWP, to
improve its skin permeability. rLMWP–EGF demonstrated sig-
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES
nificantly increased skin permeation with no loss of biological
activity or apparent toxic effect. This resulted in significantly
enhanced permeation through the intact skin around the burn
wound lesion, and also activated keratinocytes. Thus, the top-
ically applied rLMWP–EGF gel formulation significantly ac-
celerated wound reepithelialization and closure rate compared
with rEGF in a laser induced burn wound healing model. Fur-
thermore, it minimized scar and eschar formation. Microwound
formation by RF microporation in the dermis was demonstrated
to be effective for treating postburn scarring, promoting colla-
gen remodeling with minimal damage to the epidermis. How-
ever, topically applied rLMWP–EGF combined with RF micro-
poration was better for rejuvenating scarred skin with a more
normal appearance. Thus, this study suggests that rLMWP–
EGF may be a candidate therapeutic agent for treating burns.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
REFERENCES
1. Jurjus A, Atiyeh BS, Abdallah IM, Jurjus RA, Hayek SN, Jaoude
MA, Gerges A, Tohme RA. 2007. Pharmacological modulation of wound
healing in experimental burns. Burns 33:892–907.
2. Tshukudu GM, van der Walt M, Wessels Q. 2010. Comparative in
vitro study of honey based and silver based wound preparations on cell
viability. Burns 36:1036–1041.
3. Jackson DM. 1953. The diagnosis of the depth of burning. Br J Surg
40:588–596.
4. Gillitzer R, Goebeler M. 2001. Chemokines in cutaneous wound heal-
ing. J Leukoc Biol 69:513–521.
5. Werner S, Grose R. 2003. Regulation of wound healing by growth
factors and cytokines. Physiol Rev 83:835–870.
6. Goldman R. 2004. Growth factors and chronic wound healing: Past,
present, and future. Adv Skin Wound Care 17:24–35.
7. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M.
2008. Growth factors and cytokines in wound healing. Wound Repair
Regen 16:585–601.
8. Behm B, Babilas P, Landthaler M, Schreml S. 2011. Cytokines,
chemokines and growth factors in wound healing. J Eur Acad Dermatol
Venereol 26:812–820.
9. Brown GL, Curtsinger LJ, White M, Mitchell RO, Pietsch J,
Nordquist R, von Fraunhofer A, Schultz GS. 1988. Acceleration of
tensile strength of incisions treated with EGF and TGF-$. Ann Surg
208:788–794.
10. Shah M, Foreman DM, Ferguson MW. 1992. Control of scarring in
adult wounds by neutralizing antibody to transforming growth factor
beta. Lancet 339:213–214.
11. Nissen NN, Polverini PJ, Koch AE, Volin MV, Gamelli RL, DiPi-
etro LA. 1998. Vascular endothelial growth factor mediates angiogenic
activity during the proliferative phase of wound healing. Am J Pathol
152:1445–1452.
12. Smiell JM, Wieman TJ, Steed DL, Perry BH, Sampson AR, Schwab
BH. 1999. Efficacy and safety of becaplermin (recombinant human
platelet-derived growth factor-BB) in patients with nonhealing, lower
extremity diabetic ulcers: A combined analysis of four randomized stud-
ies. Wound Repair Regen 7:335–346.
13. Kawai K, Suzuki S, Tabata Y, Ikada Y, Nishimura Y. 2000. Ac-
celerated tissue regeneration through incorporation of basic fibroblast
growth factor-impregnated gelatin microspheres into artificial dermis.
Biomaterials 21:489–499.
14. Falanga V, Eaglstein WH, Bucalo B, Katz MH, Harris B, Carson
P. 1992. Topical use of human recombinant epidermal growth factor
(h-EGF) in venous ulcers. J Dermatol Surg Oncol 18:604–606.
DOI 10.1002/jps.23725
 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
15. Da Costa RM, Ribeiro Jesus FM, Aniceto C, Mendes M. 1999.
Randomized, double-blind, placebo-controlled, dose-ranging study of
granulocyte-macrophage colony stimulating factor in patients with
chronic venous leg ulcers. Wound Repair Regen 7:17–25.
16. Møller S, Jensen M, Svensson P, Skakkebaek NE. 1991. Insulin-like
growth factor 1 (IGF-1) in burn patients. Burns 17:279–281.
17. McCarthy DW, Downing MT, Brigstock DR, Luquette MH, Brown
KD, Abad MS, Besner GE. 1996. Production of heparin-binding epi-
dermal growth factor-like growth factor (Hb-EGF) at sites of thermal
injury in pediatric patients. J Invest Dermatol 106:49–56.
18. Fu X, Shen Z, Chen Y, Xie J, Guo Z, Zhang M, Sheng Z. 1998.
Randomised placebo-controlled trial of use of topical recombinant
bovine basic fibroblast growth factor for second-degree burns. Lancet
352:1661–1664.
19. Cohen S, Carpenter G. 1975. Human epidermal growth factor: Iso-
lation and chemical and biological properties. Proc Natl Acad Sci USA
72:1317.
20. Rheinwald JG, Green H. 1977. Epidermal growth factor and the
multiplication of cultured human epidermal keratinocytes. Nature
265:421.
21. Franklin JD, Lynch JB. 1979. Effects of topical applications of epi-
dermal growth factor on wound healing. Plast Reconstr Surg 64:766–
770.
22. Thornton JW, Hess CA, Cassingham V, Bartlett RH. 1981. Epider-
mal growth factor in the healing of second degree burns: A controlled
animal study. Burns 8:156.
23. Niall M, Ryan GB, O’Brien BM. 1982. The effect of epidermal
growth factor on wound healing in mice. J Surg Res 33:164–169.
24. Arturson G. 1984. Epidermal growth factor in the healing of corneal
wounds, epidermal wounds and partial-thickness scalds. Scand J Plast
Reconstr Surg 18:33–37.
25. Brown GL, Nanney LB, Griffen J, Cramer AB, Yancey JM,
Curtsinger LJ 3rd, Holtzin L, Schultz GS, Jurkiewicz MJ, Lynch JB.
1989. Enhancement of wound healing by topical treatment of epidermal
growth factor. N Engl J Med 321:76–79.
26. Chen RR, Mooney DJ. 2003. Polymeric growth factor delivery
strategies for tissue engineering. Pharm Res 20:1103–1112.
27. Hardwicke J, Schmaljohann D, Boyce D, Thomas D. 2008. Epider-
mal growth factor therapy and wound healing-past, present and future
perspectives. Surgeon 6:172–177.
28. Yang CH. 2008. Evaluation of the release rate of bioactive re-
combinant human epidermal growth factor from crosslinking collagen
sponges. J Mater Sci Mater Med 19:1433–1440.
29. Buckley A, Davidson JM, Kamerath CD, Wolt TB, Woodward SC.
1985. Sustained release of epidermal growth factor accelerates wound
repair. Proc Natl Acad Sci USA 82:7340–7344.
30. Alemdaroğlu C, Değim Z, Celebi N, Zor F, Oztürk S, Erdoğan D.
2006. An investigation on burn wound healing in rats with chitosan gel
formulation containing epidermal growth factor. Burns 2006:319–327.
31. Choi JS, Leong KW, Yoo HS. 2008. In vivo wound healing of diabetic
ulcers using eletrospun nanofibers immobilized with human epidermal
growth factor (EGF). Biomaterials 29:587–596.
32. Ulubayram K, Nur Cakar A, Korkusuz P, Ertan C, Hasirci N. 2001.
EGF containing gelatin-based wound dressing. Biomaterials 22:1345–
1356.
33. Xie Y, Upton Z, Richards S, Rizzi SC, Leavesley DI. 2011.
Hyaluronic acid: Evaluation as a potential delivery vehicle for vit-
ronectin: Growth factor complexes in wound healing applications. J
Control Release 153:225–232.
34. Johnson NR, Wang Y. 2013. Controlled delivery of heparin-biding
EGF-like growth factor yields fast and comprehensive wound healing.
J Control Release 166:124–129.
35. Cho Lee AR, Leem H, Lee J, Park KC. 2005. Reversal of silver
sulfadiazine-impaired wound healing by epidermal growth factor. Bio-
materials 26:4670–4676.
36. Breuing K, Andree C, Helo G, Slama J, Liu PY, Eriksson E. 1997.
Growth factors in the repair of partial thickness porcine skin wounds.
Plast Reconstr Surg 100:657–664.
DOI 10.1002/jps.23725
11
37. Greaves MW. 1980. Lack of effect of topically applied epidermal
growth factor (EGF) on epidermal growth in man in vivo. Clin Exp
Dermatol 5:101–103.
38. Choi JK, Jang JH, Jang WH, Kim J, Bae IH, Bae J, Park
YH, Kim BJ, Lim KM, Park JW. 2012. The effect of epider-
mal growth factor (EGF) conjugated with low-molecular-weight pro-
tamine (LMWP) on wound healing of the skin. Biomaterials 33:8579–
8590.
39. Jiang CK, Magnaldo T, Ohtsuki M, Freedberg IM, Bernerd F, Blu-
menberg M. 1993. Epidermal growth factor and transforming growth
factor " specifically induce the activation- and hyperproliferation-
associated keratins 6 and 16. Proc Natl Acad Sci USA 90:6786–
6790.
40. Kiyohara Y, Nishiguchi K, Komada F, Iwakawa S, Hirai M, Oku-
mura K. 1993. Cytoprotective effects of epidermal growth factor (EGF)
ointment containing nafamostat, a protease inhibitor, on tissue damage
at burn site rats. Bio Pharm Bull 16:1146–1149.
41. Gönül B, Erdoğan D, Ozoğul C, Koz M, Babül A, Celebi N. 1995.
Effect of EGF dosage forms on alkali burned corneal wound healing
mice. Burns 21:7–10.
42. Hashimoto K, Higashiyama S, Asada H, Hashimura E, Kobayashi
T, Sudo K, Nakagawa T, Damm D, Yoshikawa K, Taniguchi N. 1999.
Heparin-binding epidermal growth factor-like growth factor is an au-
tocrine growth factor for human keratinocytes. J Biol Chem 269:20060–
20066.
43. Shirakata Y, Komurasaki T, Toyoda H, Hanakawa Y, Yamasaki
K, Tokumaru S, Sayama K, Hashimoto K. 2000. Epiregulin, a novel
member of the epidermal growth factor family, is an autocrine
growth factor in normal human keratinocytes. J Biol Chem 275:5748–
5753.
44. Ohtake K, Maeno T, Ueda H, Natsume H, Morimoto Y. 2003. Poly-
L-arginine predominantly increases the paracellular permeability of
hydrophilic macromolecules across rabbit nasal epithelium in vitro.
Pharm Res 20:153–160.
45. Morita K, Myachi Y. 2003. Tight junctions in the skin. J Dermatol
Sci 31:81–89.
46. Thorén PE, Persson D, Esbjörner EK, Goksör M, Lincoln P, Nordén
B. 2004. Membrane binding and translocation of cell penetrating pep-
tides. Biochemistry 43:3471–3489.
47. Sharma V, Stebe K, Murphy JC, Tung L. 1996. Poloxamer 188
decreases susceptibility of artificial lipid membranes to electroporation.
Biophys J 71:3229–3241.
48. Curry DJ, Wright DA, Lee RC, Kang UJ, Frim DM. 2004. Surfactant
poloxamer 188-related decreases in inflammation and tissue damage
after experimental brain injury in rats. J Neurosurg 101:91–96.
49. Williams AC, Barry BW. 2004. Penetration enhancers. Adv Drug
Deliv Rev 56:603–618.
50. Ward RS, Saffle JR. 1995. Topical agents in burn and wound care.
Phys Ther 75:526–538.
51. Manafi A, Hashemlou A, Momeni P, Moghimi HR. 2008. Enhanced
drugs absorption through third-degree burn wound eschar. Burns
34:698–702.
52. Rockwell WB, Cohen IK, Ehrlich HP. 1989. Keloids and hyper-
trophic scars: A comprehensive review. Plast Reconstr Surg 84:827–
837.
53. Anderson RR, Parrish JA. 1981. The optics of human skin. J Invest
Dermatol 77:13–19.
54. Amadeu T, Braune A, Mandarim-de-Lacerda C, Porto LC,
Desmoulière A, Costa A. 2003. Vascularization pattern in hypertrophic
scars and keloids: A stereological analysis. Pathol Res Pract 199:469–
473.
55. Torkian BA, Yeh AT, Engel R, Sun CH, Tromberg BJ, Wong BJ.
2004. Modeling aberrant wound healing using tissue-engineered skin
constructs and multiphoton microscopy. Arch Facial Plast Surg 6:180–
187.
56. Laws RA, Finley EM, McCollough ML, Grabski WJ. 1998. Alabaster
skin after carbon dioxide laser resurfacing with histologic correlation.
Dermatol Surg 24:633–636.
Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES
12 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

57. Ayhan S, Baran CN, Yavuzer R, Latifoglu O, Cenetoglu S, Baran
NK. 1998. Combined chemical peeling and dermabrasion for deep acne
and posttraumatic scars as well as aging face. Plast Reconstr Surg
102:1238–1246.
58. Sadick NS. 2005. Combination radiofrequency and light energies:
Electro-optical synergy technology in esthetic medicine. Dermatol Surg
31:1211–1217.
59. Aust MC, Fernandes D, Kolokythas P, Kaplan HM, Vogt PM. 2008.
Percutaneous collagen induction therapy: An alternative treatment for
scars, wrinkles, and skin laxity. Plast Reconstr Surg 121:1421–1429.
60. Aust MC, Knobloch K, Reimers K, Redeker J, Ipaktchi R, Altin-
tas MA, Gohritz A, Schwaiger N, Vogt PM. 2010. Percutaneous colla-
gen induction therapy: An alternative treatment for burn scars. Burns
36:836–843.

Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.23725