Journal of Controlled Release 220 (2015) 624–630

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Nitric oxide-releasing polymer incorporated ointment for cutaneous

wound healing
Youngnam Kang a,b, Jihoon Kim a, Yeong Mi Lee a,c, Sooseok Im a,b, Hansoo Park d,⁎, Won Jong Kim a,b,c,⁎⁎
a
Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang 790-784, Republic of Korea
b
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea
c
Department of Chemistry Polymer Research Institute, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea
d
School of Integrative Engineering, Chung-Ang University, Seoul 156-75l, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: This work demonstrates the development of nitric oxide-releasing ointment and its potential on efficient wound
Received 10 June 2015 healing. Nitric oxide-releasing polymer was successfully synthesized, which is composed of biocompatible
Received in revised form 10 August 2015 Pluronic F127, branched polyethylenimine and 1-substituted diazen-1-ium-1,2-diolates. The synthesized nitric
Accepted 31 August 2015
oxide-releasing polymer was incorporated into the PEG-based ointment which not only facilitated nitric oxide
Available online 5 September 2015
release in a slow manner, but also served as a moisturizer to enhance the wound healing. As compared to control
Keywords:
groups, the nitric oxide-releasing ointment showed the accelerated wound closure with enhanced re-
Wound healing epithelialization, collagen deposition, and blood vessel formation in vivo. Therefore, this nitric oxide-based oint-
Ointment ment presents the promising potential for the efficient strategy to heal the cutaneous wound.
Nitric oxide © 2015 Elsevier B.V. All rights reserved.
N-diazeniumdiolate

1. Introduction type formulated by electrospinning of acrylonitrile-based polymer con-

Wound healing consists of complex processes involving inflamma-
tion, proliferation and remodeling [1–4]. Nitric oxide (NO), a highly
reactive free radical molecule, is produced via catalytic reaction be-
tween L-arginine substrates and NO synthases (NOSs) involving endo-
thelial (eNOS) and inducible (iNOS) nitric oxide synthases in vivo [5,
6], which is well-known to participate in each of phases of wound
healing [7–15]. In the phase of inflammation, NO mediates vasodilation
and antiplatelet effects. During the proliferative phase, NO promotes re-
epithelialization and angiogenesis [2]. In the final remodeling steps, NO
serves as an enhancer for collagen deposition [2,16,17]. Consequently,
the NO has attracted attentions as one of the efficient and biocompatible
therapeutics for wound healing.
Several synthetic NO-releasing materials have been explored for the
wound healing. Masters et al. reported poly(vinyl alcohol)-NO-modified
polymeric hydrogel for chronic wound healing, which demonstrated
improved collagen accumulation and granulation formation through
in vivo examination [18]. Blecher et al. reported that NO-loaded nano-
particle composed of silane-based sol–gel and sugar-derived glasses
promoted wound healing in mouse model with immune deficiency
[19]. Recently, Lowe et al. reported that a bandage or wound dressing
⁎ Corresponding author.
⁎⁎ Correspondence to. W.J. Kim, Center for Self-assembly and Complexity, Institute for
Basic Science (IBS), Pohang 790-784, Republic of Korea
E-mail address: wjkim@postech.ac.kr (W.J. Kim).
jugated with NO improved wound healing, though it required a labori-
ous task by daily changes of the bandages [20]. As continued to increase
in researches that develop NO-releasing materials on wound healing,
there are growing demands for the practical strategy capable of applica-
tion of general cutaneous wound healing. Among various NO delivery
systems, we focused on the pluronic-based NO delivery system which
was previously developed in our group [21]. The NO-releasing system
was composed of biocompatible Pluronic F127 for the extended NO
release by a cage recombination mechanism [22], polyethylenimine
(BPEI) as a depot of the NO donor [23], and N-diazeniumdiolates
(NONOates) [23–28] as a NO-releasing moiety. In accordance with the
reference, the pluronic-based NO-releasing polymer displayed an
increase of endothelial cell proliferation and reduction of smooth mus-
cle cell proliferation with the sustained NO release [21,29]. Moreover,
its derivative demonstrated ability to kill the bacteria [30], indicating
the potential of NO-releasing polymer for the protection of the wound
from contamination during wound healing process [31].
We supposed that the advantage of the aforementioned NO-releasing
polymer could be suitable for further application in wound healing. In
addition to that, we would like to exploit the PEG-based ointment
which has been widely utilized as a key ingredient for the commercialized
ointment. Although the supply of moisture to the wounded area is impor-
tant for the improved wound healing [32,33], the majority of researches
in the field of NO-releasing ointments have not been focused on it
[18–21]. Accordingly, we used PEG-based ointment as a storage of NO-
releasing polymers in a slow manner as well as a moisturizer to help

http://dx.doi.org/10.1016/j.jconrel.2015.08.057
0168-3659/© 2015 Elsevier B.V. All rights reserved.
 Y. Kang et al. / Journal of Controlled Release 220 (2015) 624–630
Scheme 1. Schematic illustration of overall experiment utilizing FBN ointment for the treatment of cutaneous wound.
enhance wound healing effect. In particular, we exploited its stickiness
and spreading ability for the efficient localized applications. Owing to
the combination of FBN and PEG-based ointment, our rationally designed
NO-delivery system showed sustained NO release with proper concentra-
tion and long duration for wound healing and were successfully applied
to in vivo experiment (Scheme 1). Thus, our approaches are expected to
provide a step forward to the advanced development of practical NO-
mediated ointment.
2. Materials and methods
2.1. Materials
Branched PEI (BPEI, Mw 600 Da) was obtained from Polyscience,
Inc., (Warrington, PA) and Pluronic ® F127 (F127), p-nitrophenyl
chloroformate (p-NPC), tetrahydrofuran (THF), potassium bromide,
dichloromethane (DCM), diethyl ether, dimethyl sulfoxide and
methanol were purchased from Sigma-Aldrich (St. Louis, MO). Poly-
ethylene glycols (PEG) with different molecular weight (Mw 400 Da
and 4 kDa) were obtained from Dongkook Pharmaceutical (Seoul,
Korea). Triethylamine (TEA) was obtained from Samchun Chemicals
(Pyeongtaek, Korea). Sodium methoxide (NaOMe) was purchased
from Acros Organics (Geel, Belgium). The Spectra/Por dialysis mem-
brane (MWCO 10 kDa) was purchased from Spectrum Laboratories
(Rancho Dominguez, CA). NO gas was purchased from HANA gas
(Gimhae, Korea). Argon (Ar) gas was purchased from BOC gas
(Pohang, Korea). Dulbecco's phosphate buffered saline (DPBS) was
obtained from Invitrogen (Molecular Probes Inc., Eugene, Oregon). He-
matoxylin and Eosin staining reagents were obtained from YD Diagnos-
tics (Yongin, Korea) and Duksan Reagents (Ansan, Korea), respectively.
Picro-sirius red stain kit was from Scytek Inc., (Utah, USA). CD-31 anti-
body (anti-mouse, polyclonal Ig, raised in rabbit) was from Abcam (Cam-
bridge, UK).
2.2. Synthesis and characterization of Pluronic F127-BPEI-NONOates (FBN)
FBN was synthesized with slightly modified method as reported
previously [21,30]. Briefly, to prepare activated F127, a solution of
F127 (10 g, 0.794 mmol) in anhydrous DCM was added dropwise to a
solution of p-NPC (1.29 g, 6.35 mmol) in DCM under nitrogen atmo-
sphere, and stirred for 24 h. The activated F127 was purified by precipitat-
ing the desired product with cold diethyl ether and subsequently dried in
vacuum oven. A solution of the activated F127 (2 g) in DCM was slowly
625
added to a mixture of BPEI600 (112 mg, 0.186 mmol) and TEA (1 mL)
in DCM (10 mL) at 0 °C. The reaction was carried out for 24 h under nitro-
gen atmosphere at room temperature with mild stirring. The product was
dialyzed by a Spectra/Por dialysis membrane against distilled water to
eliminate the unreacted BPEI and lyophilized to afford F127-BPEI (FB).
In order to conjugate 1-substituted diazen-1-ium-1,2-diolates called as
NONOates to it, FB (800 mg) in dry THF (3 mL) was added to a solution
of NaOMe in dry methanol (0.5 M, 20 mL), and sonicated for 5–
10 min to completely dissolve the material. The prepared solution was
then put in an in-house high-pressure NO reactor. The reactor was
first flushed with 20 psi Ar gas and then filled with NO gas at 80 psi,
which was stayed for 3 days. Then, sample solution was precipitated
with excess cold diethyl ether before the product was dried under vacu-
um in ice-cold condition and stored at −20 °C. F127, BPEI, FB and FBN
were characterized by 1H NMR in D2O using 500 MHz NMR spectrometer
(Bruker, Germany). The formation of NONOates in FBN was confirmed by
UV–vis spectrophotometry (UV 2550, Shimadzu). Additionally, FT-IR
spectra was recorded on potassium bromide pallet with FT-IR
spectroscopy (VERTEX70 FT-IR spectrophotometer, Bruker Optics) to
confirm the characteristic peaks of NONOates of FBN.
2.3. Preparation of FBN-containing PEG-based ointment
PEG-based ointment was prepared according to the protocol of
Dongkook Pharmaceutical. PEG 400 Da (80 mg) and PEG 4 kDa
(40 mg) were mixed at the ratio of 2:1. The mixture was then heated
over 60 °C, turning to a clear solution state. The prepared FBN (1 mg)
was added to the solution before cooling down at room temperature
to form gel.
Table 1
NO release properties of FBN and FBN/PEG.a
Materials [NO]tb tdc [NO]md tme t1/2f
FBN 9.4 0.5 18.9 3.4 5.9
FBN/PEG 95.8 4.1 69.7 4.0 23.4
a
Measured in DPBS at 37 °C by NO Analyzer (NOA 280i chemiluminescence).
b
[NO]t, The number of moles of NO release per mg (nmol).
c
td, duration of NO release above 1 pmol mg−1 (h).
d
[NO]m, maximum instantaneous concentration of NO release (pmol mg−1).
e
tm, time required to reach [NO]m (min).
f
t1/2, half-life of NO release (min).
626 Y. Kang et al. / Journal of Controlled Release 220 (2015) 624–630
Fig. 1. Sol–gel transition of PEG-based ointment (FBN/PEG) observed during heating and cooling process. (A) Just mixing PEG400 with PEG4000 prior to heating (B) Heating the mixture up
to 68 °C (C) Cooling at room temperature after adding FBN/PEG.
2.4. Measurement of NO release
Quantitative NO release was measured by chemiluminescence using
an NO analyzer (Sievers 280i Nitric Oxide Analyzer, NOA, GE analytical
instruments, USA). Chemiluminescence measurement was used to
evaluate the kinetics of NO release under physiological condition; in a
DPBS solution, pH 7.4, at 37 °C under Ar gas flow. Each 1 mg of FBN and
FBN/PEG in 40 mL DPBS was used to investigate their NO release profile.
Various parameters such as [NO]t (the total number of moles of NO
release per mg), td (duration of NO until NO release is finished), [NO]m
(maximum instantaneous concentration of NO release), tm (time required
to reach [NO]m), and t1/2 (half-life of NO release) were evaluated by NO
release profile.
2.5. In vivo wound healing assay
The POSTECH Biotech Center Ethics Committee approved all of the
animal experiments in this study. Five to six week-old female BALB/C-
nu/nu mice were used and randomly grouped into four (control, PEG,
FB/PEG and FBN/PEG) comprising seven animals each. The hair on the
back of each mouse was shaved on a day before the surgery. Single
punch biopsies (5 mm Punch Biopsy; Miltex, York, Pennsylvania) were
performed to create full-thickness cutaneous wound with 5 mm size
in diameter. NO is known for inhibition of platelet aggregation [2,34];
thus, treatment was started 1 day after wounding to provide appropriate
time for fibrin clot formation. The 120 μL ointment (PEG, FB/PEG and
FBN/PEG) was prepared on the day of treating. Then, the ointment
was applied to the wounds every other day. On days 0, 3, 9 and 13,
wounds were digitally photographed by COOLPIX S6500 camera
(Nikon). Wound area was quantified using an image analysis program
(ImageJ®, NIH). One mouse per group was sacrificed each alternate day
Fig. 2. NO release from FBN and FBN/PEG monitored by NO analyzer. Inset represents the
cumulative NO release.
until 11 day and wound and surrounding tissues were collected for
histological evaluation. Remaining mice were all sacrificed on day 13.
The wound closure was expressed as the percentage area of the initial
wound size [35].
2.6. Histology and immunohistochemistry
2.6.1. Hematoxylin and eosin (H&E) and Sirius red staining
The wound and surrounding tissue (~5 mm) were excised and fixed
in 10% neutral buffered formalin for 48 h (Sigma-Aldrich, St. Louis, MO)
and embedded in paraffin and sequentially sectioned at 4 μm using a
Finesse ME microtome (Thermo Fisher Scientific). Skin sections were
stained with hematoxylin and eosin (H&E) for the assessment of granu-
lation tissue formation and wound maturity and with Sirius red staining
to study the extent of collagen deposition in healed tissue during the
course of wound healing. All histological analyses were performed on
at least 4 wounds per group and images presented are representative of
all replicates. Images of entire sections were achieved by a microscopy
(Nikon eclipse 80i, USA) with 4× magnification located at the Pohang
Center for Evaluation of Biomaterials (Pohang Technopark).
2.6.2. CD 31 immunofluorescent staining
Skin sections embedded in paraffin were cut at 4 μm thickness. An
antibody directed against the murine endothelial cell surface marker
(CD-31) was used to investigate the extent of endothelial cell coloniza-
tion in the wound sections. After treating blocking solution (5% (w/v)
BSA in distilled water) to avoid non-specific binding sites, the primary
antibody was applied for 1.5 h at room temperature, followed by wash-
ing several times with PBS for 20 min. Secondary antibody conjugated to
goat anti-rabbit IgG-FITC was then treated for 1 h to visualize the
antigen. Finally, sections on coverslip were mounted in DAPI mounting
medium to visualize the cell nuclei and stored in the dark at 4 °C. Fluores-
cent images were taken by using confocal laser scanning microscope
(Modified Zeiss Axio Observer, Z1 epi-fluorescence microscope) with
10× magnification. The images were representative ones of local areas
of wound sections stained with CD 31 uniformly distant from the epider-
mis in all groups (Fig. 7).
2.7. Statistical analysis
All experiments were repeated at least three times and each condi-
tion was analyzed in triplicate. Statistical analysis was performed
using GraphPad Prism 5.0 software (GraphPad Software, Inc., California,
USA). The values were expressed as means ± standard error of the
mean (S.E.M.). One-way ANOVA followed by Tukey's post-hoc was
used to discern the statistical difference between groups.
3. Results and discussion
3.1. Synthesis and characterization of Pluronic F127-BPEI-NONOates (FBN)
FBN was prepared according to a previously described protocol
(Supplementary data, Scheme S1) [21,30]. In brief, biocompatible
Pluronic F127 was conjugated with BPEI via p-NPC chemistry to afford
 Y. Kang et al. / Journal of Controlled Release 220 (2015) 624–630
Fig. 3. Representative images of wounds of four test groups; control, PEG, FB/PEG and FBN/PEG over time in each group.
FB. In particular, the low Mw BPEI600 was exploited for developing FB to
reduce the cytotoxicity and provide the reservoir for NONOates. FBN
was synthesized by conjugating NONOates to the secondary amine moi-
ety of BPEI from FB. The structural characteristics of FBN were confirmed
by 1H NMR, UV/vis spectroscopy and FT-IR (Supplementary data,
Figs. S1, S2). In 1H NMR, FBN showed a downfield shift of proton signals
of methylene group neighboring to secondary amine of BPEI from
2.7–2.8 ppm to 3.1 ppm due to electron-withdrawing effect of the
NONOates group (Supplementary data, Fig. S1) [21]. The UV/vis ab-
sorption spectra showed strong peak for NONOates in FBN at about
250 nm [30]. Meanwhile, F127 and FB did not show any specific absorp-
tion peak at 250 nm. In accordance with the previous reports [30] the
characteristic peaks of NONOates in FBN were observed in FR-IR spec-
troscopy; N-O stretch (1390–1410 cm), NO vibration (1735 cm), N =
N stretch (1620–1630 cm), N-N stretch (1068 cm, 1480–1540 cm)
(Supplementary data, Fig. S2).
3.2. NO release profile of FBN and FBN/PEG
Previously, NO release was monitored by Griess assay [21], while NO
release from FBN was measured by chemiluminescence NO analyzer for
real-time detection in the present study. NO release from 1 mg of FBN
was evaluated in the physiological condition (pH 7.4 DPBS solution at
37 °C), and it liberated overall 9.4 nmol NO in 0.5 h. The maximum
instantaneous concentration of NO release was recorded as 18.9 pmol/s
at 3.4 min and the half-life of NO release was found to be 5.9 min
(Table 1). For the successful wound healing process, we expected that vis-
cous PEG has the cage recombination effect which sustains the release of
NO in the gel by physically preventing rapid contact between NONOates
functional groups and water [22]. Along with that, because PEG obtained
from Dongkook Pharmaceutical has a slightly acidic pH (b pH 5) and the
NO release from NONOates is based on the acid-catalyzed reaction, it was
also expected that the acidity of PEG triggers higher values of NO release
627
[18,36–38]. Therefore, the FBN/PEG was prepared by mixing FBN with
PEG when PEG undergoes the transition from sol to gel during cooling
process (Fig. 1). Especially, it is important to note that FBN was needed
to be stored at −4 °C prior to mixing with PEG to prevent instantaneous
NO release. In practice, the FBN/PEG exhibited total 95.8 nmol NO release
and 4.1 h duration (Fig. 2). As compared to FBN, FBN/PEG yielded about
10-fold enhancement in total amount of released NO and around 8-fold
increase in overall duration (Table 1). Therefore, it was demonstrated
that the PEG-based ointment has ability to control the release of NO in a
sustainable manner, which raised sense of expectancy for its potential
on successful wound healing.
3.3. Evaluation of mouse wound healing
Wound healing effects of PEG, FB/PEG and FBN/PEG were evaluated
by calculating the wound closure (%) through the measurement of the
wound area (px2) on the day of surgery and on days 3, 9 and 13 after
wounding. Digital images of representative wounds of each group
from days 0, 3, 9 and 13 are shown in Fig. 3. On day 3, no significant
differences were found among the groups (Fig. 4A). Nevertheless,
PEG-containing groups (PEG, FB/PEG and FBN/PEG) showed rather
slight increase of wound closure possibly because PEG provides a
moist environment around wounds (Fig. 4B) [39]. However, at day 9,
FBN/PEG showed a clear accelerated wound healing compared to that
of other groups with significantly greater differences (p b 0.005 for no
treatment and PEG and p b 0.05 for FB/PEG). The wound area of mice
treated with FBN/PEG was decreased by 80.5% relative to day 0, com-
pared to 63.8% in mice with control, 67.8% in PEG-treated mice and
67.9% in FB/PEG-treated mice (Fig. 4C). On the 13th of post-wounding
day, total wound closure was appreciated the most in mice treated
with FBN/PEG by 86.5%. Therefore, these data suggest that NO applica-
tion using FBN/PEG accelerates wound closure especially during the
early proliferative healing process from day 3 to day 9 [2–4], resulting
628 Y. Kang et al. / Journal of Controlled Release 220 (2015) 624–630
Fig. 4. Effects of FBN/PEG on wound closure. (A) Healing progression for days 0–13. Values
are presented as a percentage of the wound area compared to day 0. (B) Percentage
wound closure on day 3 after wounding. (C) Percentage wound closure on day 9 after
wounding. (mean ± SEM). (**P b 0.05; ***P b 0.0001).
in better wound healing effect compared to the other groups. No clini-
cally significant weight loss or evidence of infection in any mouse was
appreciated throughout the study period.
Fig. 5. Hematoxylin and eosin (H&E)-stained sections of the wound sites of control or treated with PEG, FB/PEG and FBN/PEG on days 3, 9, and 13, respectively. Scale bar represents 500 μm.
3.4. Histological analysis
3.4.1. Re-epithelialization and granulation tissue formation
To confirm re-epithelialization and granulation tissue formation,
wound skin sections were stained with H&E. This histological examina-
tion demonstrated general morphology of skin layers during the process
of healing (Fig. 5). On day 3, all groups showed wound scabs which keep
more blood and other fluids from flowing out and prevent wounds from
attacking any pathogens outside [2–4]. This represented that early
inflammatory healing process was occurred in all groups on day 3.
Additionally, it was shown in all groups that the borders between epider-
mis and dermis around were ambiguous and the alignment of dermis
loosely maintained since this inflammatory phase activates platelet
clotting and macrophage generating rather than re-epithelialization and
dermal remodeling [3–4]. As expected, on day 9, granulation tissue
formation was significantly increased at FBN/PEG group compared to
other groups. It was clearly visible that in FBN/PEG group, the epidermal
and subepidermal layers were well organized and covered by mature
epithelium. On day 13th post-wounding, all groups presented granulation
tissue formation and dermal remodeling. In particular, FBN/PEG group
showed complete re-epithelialization of the wound with well differenti-
ated epithelium and reduced area of scab owing to fast dermal remodel-
ing comparing to the other groups. Indeed, NO has been known to
stimulate the proliferation of keratinocyte, endothelial cell and fibroblast
by modulating cytokines, which involved in the beginning of the prolifer-
ative phase of wound healing [40–42]. Thus, the same stimulating effects
were expected in the exogenous administration of NO from FBN/PEG,
exerting these actions after diffusing to the tissue of the wound.
3.4.2. Collagen deposition
To evaluate a collagen deposition with the help of NO, sections of skin
wounds were stained with Sirius red, which revealed the extent of colla-
gen deposition in healed tissue (Fig. 6). The images of the sectioned areas
are well corresponded to those stained with H&E. On day 3, all groups
presented a loose arrangement of collagen from intensity of Sirius red
staining (pink). On day 9, in comparison with other groups, FBN/PEG
treatment showed higher collagen content under subepidermis by show-
ing more packed color of collagen (red). On day 13th post-wounding, a
higher intensity of collagen and dense alignment were observed in FBN/
PEG-treated group compared to other groups. This finding might be
attributed to the fact that NO increases collagen content of wound by
modulating TGF-β1, a transforming growth factor that regulates prolifer-
ation, differentiation and other functions in many cell types [43,44].
3.4.3. Angiogenesis
Angiogenesis is an essential factor of normal wound repair, and NO
appears to serve as a critical component in this process [45,46]. To
investigate the angiogenesis during wound healing process, we focused
on the epidermis where endothelial cells are dominated around the
 Y. Kang et al. / Journal of Controlled Release 220 (2015) 624–630
Fig. 6. Sirius red-stained sections of the wound sites of control or treated with PEG, FB/PEG and FBN/PEG on days 3, 9, and 13, respectively. Scale bar represents 500 μm.
wounded area. Immunofluorescence staining for endothelial cells with
CD 31 indicated the vascularization involved in angiogenesis [47]. The
evident differences of angiogenesis were observed on day 9 that CD
31-experssed endothelial cells (green) were abundantly detected in
the FBN/PEG-treated group compared with other groups (Fig. 7). This
result demonstrated that NO from FBN/PEG is attributed to angiogene-
sis. Meanwhile, at day 13, there was a significant paucity of CD 31
expressed cells in the wounds treated with FBN/PEG whereas other
groups presented readily recognizable CD 31 expressed cells (Fig. S3).
This result was meant the fact that on day 13 FBN/PEG-treated group
showed almost complete wound healing, resulting in less angiogenesis.
On the other hand, other groups whose healing process was still ongoing
showed more angiogenesis.
Taking all these considerations into account, FBN/PEG enhanced
higher re-epithelialization and collagen deposition until the final day
13 and induced a significant angiogenesis on day 9 because of the
underlying mechanism of NO involving in early proliferative phase of
wound healing. Nevertheless, there are still several hurdles for the prac-
tical applications of FBN/PEG, such as potential cytotoxicity of BPEI and
long preparing process of NO-releasing ointments. Therefore, the efforts
Fig. 7. Immunofluorescent CD31 (green) staining of day 9 wound sections. Nucleus was
stained with DAPI (blue). Scale bar represents 100 μm.
629
to overcome these difficulties would pave the way for the clinical appli-
cation of NO-releasing ointments in wound healing.
4. Conclusion
In this study, we have developed a biocompatible NO-releasing oint-
ment composed of PEG and NONOates-containing polymers for enhanced
wound healing. We demonstrated that NO was released in a sustainable
manner from FBN/PEG improved the healing activity in the early phase
of wound healing compared to control, PEG alone and FB/PEG. In vivo re-
sults demonstrated that the enhanced wound healing effect was attribut-
ed to higher re-epithelialization, granulation formation, collagen
deposition and improved angiogenesis. Taken together, these results sug-
gest that NO-releasing polymer incorporated ointment could be exploited
to promote healing of cutaneous wounds.
Acknowledgments
This work was supported by the Research Center Program of IBS
(Institute for Basic Science) in Korea (CA1203-02). The authors would
like to thank Dongkook Pharmaceutical for providing PEG ointments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2015.08.057.
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