Synthesis and physicochemical properties of new tripodal amphiphiles bearing fatty acids as a hydrophobic group

Article abstract of DOI:10.1016/j.bmcl.2013.01.052

Saturated fatty acids (FA) were grafted using tyrosine as a spacer group to the cyclotriphosphazene ring along with equimolar hydrophilic methoxy poly(ethylene glycol) (MPEG) in cis-nongeminal way. Seven new cyclotriphosphazene amphiphiles were prepared from combinations of hydrophilic MPEGs with different molecular weights of 350, 550, 750 and 1000 and four different fatty acids of different hydrophobicity including lauric, myristic, palmitic and stearic acids. These steric amphiphiles bearing fatty acids as a hydrophobic group were found to form more stable micelles with very low critical micelle concentrations (CMC) (2.95-7.80 mg/L) compared with oligopeptide analogues, and their highly hydrophobic core environment is unique and potentially useful for various biomedical applications.

Full text of DOI:10.1016/j.bmcl.2013.01.052

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1763–1767
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and physicochemical properties of new tripodal
amphiphiles bearing fatty acids as a hydrophobic group
b,
Prakash G. Avaji a, Vithal B. Jadhav b, Jin Xin Cui b, Yong Joo Jun b, Hwa Jeong Lee a, , Youn Soo Sohn
⇑
⇑
a Center for Cell Signaling & Drug Discovery Research, Division of Life and Pharmaceutical Sciences & College of Pharmacy, Ewha Womans University, 11-1 Daehyun-Dong,
Seodaemun-Ku, Seoul 120-750, Republic of Korea
b C & Pharm, Rm 263 Science Building B, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Ku, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Saturated fatty acids (FA) were grafted using tyrosine as a spacer group to the cyclotriphosphazene ring
along with equimolar hydrophilic methoxy poly(ethylene glycol) (MPEG) in cis-nongeminal way. Seven
new cyclotriphosphazene amphiphiles were prepared from combinations of hydrophilic MPEGs with dif-
ferent molecular weights of 350, 550, 750 and 1000 and four different fatty acids of different hydropho-
bicity including lauric, myristic, palmitic and stearic acids. These steric amphiphiles bearing fatty acids as
a hydrophobic group were found to form more stable micelles with very low critical micelle concentra-
tions (CMC) (2.95–7.80 mg/L) compared with oligopeptide analogues, and their highly hydrophobic core
environment is unique and potentially useful for various biomedical applications.
Received 30 October 2012
Revised 3 January 2013
Accepted 15 January 2013
Available online 24 January 2013
Keywords:
Cyclotriphosphazene
Tripodal amphiphile
Micelle
Ó 2013 Elsevier Ltd. All rights reserved.
Drug delivery
Fatty acid
Polymeric micelles have been extensively studied for last dec-
ades aiming at clinical applications as drug delivery systems for
polymer therapeutics.1,2 Polymeric micelles exhibit various advan-
tageous properties associated with solubilization of hydrophobic
drugs, potential tumor targeting by enhanced permeability and
retention (EPR) effect, controlled drug release, and possible long
blood circulation.3–6 However, success of clinical applications of
polymeric micelles has been limited, because it is not easy to de-
sign a polymeric micelle that can satisfy simultaneously all the
clinical requirements including stability, selectivity and releasing
kinetics of the drug-loaded micelle as well as biodegradability
and nontoxicity of the polymeric micelle itself.
Polymeric micelles are one of the soft nanostructures self-
assembled from amphiphilic block copolymers or graft copoly-
mers, but most of the spherical micelles are composed of linear
di- or tri-block copolymers constituted of hydrophilic and hydro-
phobic segments. Intermolecular hydrophobic interaction among
the hydrophobic segments of the copolymer molecules (unimer)
in aqueous solution lead to formation of the hydrophobic core with
the hydrophilic shell of spherical micelles. Typical size of
polymeric micelles is between 20 and 100 nm in diameter and
their critical micelle concentration (CMC) is in the range of
10–100 mg/L depending on the kind and molecular weight of the
block copolymers.3,6 The hydrophobic core of polymeric micelles
can accommodate hydrophobic drugs, which therefore can be sol-
ubilized in aqueous solution.
However, one of the most essential properties of polymeric
micelles required for clinical application is the stability of drug-
loaded polymeric micelles rather than that of the polymeric
micelles themselves. For example, most of the conventional
di-block copolymers including PEG-b-PLA,7 PEG-b-CL,8 and PEO-
b-PBO9 are known to form stable micelles in aqueous solution,
but when loaded with docetaxel, these polymeric micelles cannot
hold the drug, which precipitates within 24 h after drug loading.
In fact, it is known that important physicochemical properties such
as stability, loading capacity and releasing kinetics of drug-loaded
polymeric micelles are greatly dependent upon the properties both
of the micelle core and the drug molecules loaded.4,10
Therefore, in order to overcome such limited properties attrib-
uted from the conventional linear block copolymers and to expand
the properties of the micelle core environment, it is urgent to de-
velop new types of polymeric micelles. In this context, we have re-
cently developed a new class of tripodal amphiphiles of a general
formula [N = P(PEG)(OPE)]3 bearing poly(ethylene glycol) (PEG)
as a hydrophilic group and oligopeptide (OPE) as a hydrophobic
group.11 These novel steric amphiphiles were found to self-assem-
ble in aqueous solution into very stable spherical micelles with a
mean diameter of 7.4 to 13.9 nm,12 and the resultant hydrophobic
core composed of multifunctional oligopeptide groups provides a
unique polar hydrophobic core environment, which can stably
⇑
Corresponding authors. Tel.: +82 2 3277 3409; fax: +82 2 3277 2851 (H.J.L.);
tel.: +82 2 3277 4549; fax: +82 2 3277 3419 (Y.S.S.).
E-mail addresses: hwalee@ewha.ac.kr (H.J. Lee), yssohn@ewha.ac.kr (Y.S. Sohn).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.052

1764
P. G. Avaji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1763–1767
encapsulate docetaxel.10 In this study we have found that extre-
mely hydrophobic lipid core environment can be created to accom-
modate nonpolar hydrophobic drugs in the micelle core by
introducing saturated fatty acids instead of oligopeptides into the
cyclic phosphazene template as a hydrophobic group. Herein we
report synthesis and properties of steric amphiphiles bearing equi-
molar methoxy poly(ethylene glycol) and saturated fatty acid.
Since saturated fatty acids have only one functional group, car-
boxylic acid, which is known to lead to phosphazene ring cleavage
reaction,13 it was necessary to employ a spacer group with two
functional groups such as tyrosine to link fatty acid to the phos-
phazene ring. Among the saturated fatty acid ethyl esters,
CH3(CH2)nCOOEt, lauric (n = 10), myristic (n = 12), palmitic
(n = 14), and stearic (n = 16) acids were selected considering their
hydrophobicity defined as logP where P = [solute]n-octanol/[sol-
ute]water14 and the hydrophilicity of methoxy poly(ethylene glycol)
(MPEG) to be employed. Thus, in order to link fatty acid to the cyc-
lic phosphazene ring, we have prepared in the first step tyrosine-
fatty acid conjugates according to the following reaction Scheme 1
(Supplementary data). Fatty acids were easily coupled with tyro-
sine through amide bond using N-hydroxysuccinimide (NHS) and
N,N0-dicyclohexylcarbodiimide (DCC) as coupling agents. The
resultant tyrosine-fatty acid conjugates were transformed to so-
dium salts for nucleophilic substitution with the chlorine atoms
of the cyclic phosphazene trimer.
In order to obtain pure tripodal cyclotriphosphazene amphi-
philes, it is important to perform the initial PEGylation reaction
of the cyclotriphosphazene ring at low temperature (<ꢀ20 °C) so
that the first three chlorine atoms of hexachlorocyclotriphospha-
zene can be substituted with PEG in cis-nongeminal way. Then
the tyrosine-fatty acid conjugates (Tyr-FA) were grafted by substi-
tution with the remaining three chlorine atoms of the cyclotri-
phosphazene ring according to the following reaction Scheme 2
(Supplementary data). The 1H and 31P NMR spectra measured
using Varian 500 MHz NMR spectrometer are illustrated in Fig-
ure 1. In particular, it is clearly seen from the single peak of phos-
phorus resonance that both of hydrophilic and hydrophobic side
groups are grafted in cis-nongeminal way to form a tripodal steric
amphiphile as shown in the Scheme.
However, MPEG550 and MPEG750 produced water soluble deriva-
tives with both Tyr-Lau and Tyr-Myr conjugates. More hydropho-
bic Tyr-Pal and Tyr-Ste conjugates gave rise to water soluble
products only with more hydrophilic MPEG1000. Thus we have
prepared from appropriate combinations of MPEGs and Tyr-FA
conjugates shown in the table seven soluble amphiphilic cyclotri-
phosphazenes, which were clearly characterized by means of ele-
mental analysis, 1H and 31P NMR, dynamic light scattering (DLS),
critical micelle concentration (CMC), etc. (Supplementary data).
All these amphiphilic cyclotriphosphazene products were obtained
in little sticky white solids, which are very soluble in water and po-
lar organic solvents.
The morphology of the present trimer amphiphiles bearing fatty
acids was found to be spherical micelles, as shown in the TEM im-
age of [NP(MPEG1000)(Tyr-Ste)]3 measured by JEM-2100F15 and
illustrated in Figure 2. Also the particle size distributions of the
present fatty acid grafted amphiphiles were measured for 0.5%
aqueous solution by dynamic light scattering (DLS) method using
Malvern Zetasizer (Nano-ZS), and the representative results are
displayed in Figure 3. Thus the fatty acid grafted amphiphiles exhi-
bit the same morphology as the oligopeptide analogues but have
shown quite different behavior in aqueous solution. For example,
we have shown that, in case of oligopeptide analogues,16 the mor-
phology of the trimer amphiphiles is determined mainly by the
hydrophobicity of the oligopeptide employed (logPolig). Thus if
logPolig is in the range of 0–1, the trimer amphiphiles initially
form spherical micelles of 10–20 nm in aqueous solution but
slowly reassemble into larger double layered polymersomes of
100–1000 nm in diameter. However, if logPolig is larger than 1,
the initially formed micelles of 7–8 nm in diameter remain un-
changed in morphology. In contrast to such a variable morphology
of the trimer amphiphiles bearing oligopeptides, the trimeric ana-
logues of tyrosine-fatty acid conjugates with very high hydropho-
bicity (logPTyr-FA = 5.26–7.84) were found to form only highly
stable micelles with extremely law CMC values in aqueous solution
as will be shown later. Such a different behavior in morphology
seems to be attributed to the difference in the chemical nature of
the two hydrophobic groups. In other words, oligopeptides have
many functional groups such as amine and carbonyl groups around
the peptide backbone, which can afford partially polarized micelle
core environments while fatty acids are mainly composed of ali-
phatic hydrocarbon chains without polar groups. Such a difference
in the chemical environment of the micelle cores composed of the
trimer amphiphiles bearing fatty acids and oligopeptides is ex-
pected to affect the physicochemical properties such as micelle
size and stability, thermal sensitivity, drug loading capacity, and
drug releasing kinetics of the micelles.
We have attempted to prepare water soluble analogues of
amphiphilic cyclotriphosphazenes bearing fatty acids by different
combinations from four MPEGs of different hydrophilicity and four
fatty acids of different hydrophobicity shown in Table 1. We have
found that water insoluble products are resulted when the overall
hydrophobicity (logPsum), that is, the sum of the hydrophobicity of
the hydrophobic group, Tyr-FA (logPTyr-FA) and that of the hydro-
philic group MPEG (logPMPEG) is larger than 4.5. For example,
MPEG350 (logP = ꢀ1.42) gave a water soluble cyclotriphosphazene
product with Tyr-Lau conjugate (logP = 5.26) but insoluble prod-
ucts with Tyr-Myr conjugate (logP = 6.17) and other more hydro-
phobic tyrosine-fatty acid conjugates (Tyr-FA) in the table.
In order to examine the physicochemical properties of the
fatty acid grafted cyclotriphosphazene amphiphiles as new drug
delivery systems, in addition to the DLS measurements above-
mentioned, we have measured their lower critical solution
O
H
N
O
R
H
N
O
R
n
NaH
1. DCC, NHS, EtOAc, 12h, rt
O
O
OH
n
O
THF, rt, 12h
O
ONa
n
2. TEEH, TEA, CHCl3, 24h, rt
OH
Tyr-FA
n = 10 = Lauric acid
n = 12 = Myristic acid
n = 14 = Palmitic acid
n = 16 = Stearic acid
Na
Tyr-FA
R = C2H5
Scheme 1. Synthetic route to conjugation of fatty acids to a spacer group tyrosine.

P. G. Avaji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1763–1767
1765
Cl
O
Cl
O
m
O
Cl
P
m
P
N
NaO
N
N
P
N
P
N
3
3
-65oC, THF, 6h
Cl
Cl
Cl
P
Cl
Cl
Cl
O
m
O
MPEG
Na
rt, THF, 24h
Tyr-FA
Cyclotriphosphazene
Tyrosine spacer
Fatty acid
N
P
3
Tyr-FA
Scheme 2. Synthetic route to amphiphilic cyclotriphosphazenes bearing fatty acids as a hydrophobic group.
the fatty acid analogues measured by the fluorescence probe tech-
nique (Fig. 4) displayed much lower values of 2.95–7.80 mg/L com-
pared with 7–20 mg/L of the oligopeptide analogues, which
indicates that the micelle stability of the present fatty acid ana-
logues is much higher compared with that of the oligopeptide ana-
logues. To our knowledge, the CMC values of the present fatty acid
grafted cyclotriphosphazenes are among the lowest of the known
polymeric micelles.
The present trimer amphiphiles bearing fatty acids are thermo-
sensitive like the oligopeptide analogues. As seen in Table 2, the
LCSTs of all the fatty acid analogues are abnormally high (39.4–
82.2 °C) compared with those of the oligopeptide analogues.12 It
is generally known that the LCST of an amphiphile is lowered if
the hydrophobicity of the hydrophobic group is increased for the
same hydrophilic group of the amphiphile due to easier aggrega-
tion of the micelle particles for precipitation by stronger intermo-
lecular hydrophobic interactions among the unimers.12,19 On the
other hand, if the hydrophilicity of the hydrophilic group is in-
creased for the same hydrophobic group, the LCST of the amphil-
phile is increased because of stronger intermolecular interaction
between the hydrophilic group of the micelle corona and the sol-
vent water molecules, which inhibit aggregation of the micelle par-
ticles. According to this rule, the LCSTs of all the fatty acid
analogues in Table 2 should be lower than those of the oligopep-
tide analogues with the same hydrophilic group MPEG because
of much higher hydrophobicity of the fatty acids compared with
the oligopeptide employed. However, comparing directly two rep-
resentative analogues bearing the same hydrophilic group
MPEG550 but different hydrophobic groups Tyr-Lau (logP = 5.26)
and (GlyPheLeu)2Et (logP = 1.11), the LCST of the fatty acid
analogue [NP(MPEG550)(Tyr-Lau)]3 appears at much higher
72.6 °C in water than that of the hexapeptide analogue
[NP(MPEG550)(GlyPheLeu)2Et]3 at 48 °C.12 Similarly, other fatty
acid analogues exhibit higher LCSTs compared with the oligopep-
tide analogues bearing the same MPEG. Such a surprising result
seems to be attributed to the different chemical nature of the
two hydrophobic groups. It may be conjectured that if the hydro-
phobic group of the amphiphile is too hydrophobic like fatty acids,
strong intermolecular hydrophobic interaction within the micelle
core may hamper the dynamic interaction among micelles to pre-
cipitate. As seen in Table 2, the LCSTs of the fatty acid analogues
range from body temperature to over 80 °C, and all the fatty acid
analogues except for trimer 1 are potentially useful for intrave-
nously injectable drug carrier.
Figure 1. 31P NMR (a) and 1H NMR (b) spectra of amphiphilic cyclotriphosphazene
1.
temperature (LCST) by cloud point method17 for 5% polymer solu-
tion in distilled water and PBS contained in a glass capillary im-
mersed in an oil bath. We have also measured their critical
micelle concentration by the fluorescence probe technique using
pyrene.18 All the measured data of the present cyclotriphospha-
zene amphiphiles are listed in Table 2.
The micelle size of the trimer amphiphiles bearing oligopeptide
was reported to be in a wide range of 7.4–27.8 nm in diameter
depending on their chemical structure and hydrophobicity of the
hydrophobic oligopeptide,12 but the present fatty acid analogues
exhibit quite smaller size of 5.47–10.3 nm in diameter with nar-
rower size distribution (Fig. 3) probably due to their much higher
hydrophobicity. Also the critical micelle concentration (CMC) of
Finally, the drug loading capacity and the stability of drug-
loaded micelles are more critically dependent on the nature of

1766
P. G. Avaji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1763–1767
Table 1
Hydrophobicity of tyrosine-fatty acid conjugates and methoxy poly(ethylene glycol)s employed as side groups
Hydrophobic and hydrophilic groupsa employed as side groups
Empirical formula
C23H37NO4
Mol. Wt.
Hydrophobicity (logP)b
Tyrosine-lauramide (Tyr-Lau)
Tyrosine-myristamide (Tyr-Myr)
Tyrosine-palmitamide (Tyr-Pal)
Tyrosine-stearamide (Tyr-Ste)
MPEG350
MPEG550
MPEG750
MPEG1000
391.5
419.6
447.6
475.7
340.2
560.5
736.4
1000.5
5.26
6.17
7.00
C
C
C
25H41NO4
27H45NO4
29H49NO4
7.84
C15H32O8
ꢀ1.42
ꢀ2.24
ꢀ2.80
ꢀ4.00
C
C
C
25H52O13
33H68O17
45H92O23
a
Empirical formula of methoxy poly(ethylene glycol) was estimated from the general formula of CH3O(CH2CH2O)nH assuming n = 7 for MPEG350; n = 12 for MPEG550;
n = 16 for MPEG750; n = 22 for MPEG1000.
Hydrophobicity is defined as logP where P = [solute]n-octanol/[solute]water
b
14
.
20
18
16
14
12
10
8
(a)
6
4
2
0
0.1
1
10
100
1000
Size (nm)
Figure 2. Transmission electron microscopic image of [NP(MPEG1000)(Tyr-Ste)]3.
16
14
12
10
8
(b)
the micelle core because of the intermolecular interactions be-
tween the drug molecules and micelle core environment as were
illustrated in recent reports. For example, adriamycin (ADR) could
not be properly loaded into the micelle composed of poly(ethylene
glycol)-poly(b-benzyl L-aspartate) block copolymers (PEG-PBLA)
because of poor affinity of the hydrophobic PBLA segment with
ADR.4 As early mentioned in the introductory section in this Letter,
docetaxel can be solubilized by micelle-encapsulation using the
conventional block copolymers,7,8 but their micelle cores cannot
hold docetaxel probably due to weak intermolecular interaction
between the docetaxel molecules and micelle core environment.
However, we have shown that our cyclotriphosphazene amphi-
phile bearing hydrophilic MPEG750 and hydrophobic hexapeptide
represented as [NP(MPEG750)(GlyPheLeu)Et]3 (CP750) has not
only a large loading capacity of docetaxel (>25%) but also high sta-
bility of drug-loaded micelles10 probably because of the locally
polarized core environment composed of the hexapeptide groups
as above-mentioned. Interestingly, we have most recently found
that the present fatty acid analogues [NP(MPEG750)(Tyr-FA)Et]3
can solubilize docetaxel by micelle-encapsulation like the hexa-
peptide analogue CP750 but cannot hold the drug molecules in
their micelle core unlike CP750, and docetaxel precipitated in a
few days.
On the contrary, if the drug to be formulated by micelle-encap-
sulation changes from docetaxel (logP = 2.59) to a highly hydro-
phobic drug like propofol (2,6-diisopropyl phenol) (logP = 4.11),
the situation may be reversed. Propofol is currently one of the most
widely used anesthetic agent which is marketed as 1% propofol for-
mulated in oil emulsion.20 We have tested loading capacity and
stability of propofol micelle-encapsulated by the present fatty acid
grafted amphiphile [NP(MPEG750)(Tyr-Lau)Et]3 and the hexapep-
tide analogue CP750 for comparison. Propofol could be loaded into
CP750 up to 10% of the carrier by our solvent evaporation meth-
6
4
2
0
0.1
1
10
100
1000
Size (nm)
Figure 3. The particle size distributions of amphiphilic cyclotriphosphazenes 1 (a)
and 7 (b).
od,10 but when the propofol formulated by micelle-encapsulation
using CP750 was attempted to dissolve up to 1% propofol in dis-
tilled water, no clear solution was obtained due to precipitation
of propofol. In other words, the propofol loaded into CP750 was
not stable. On the other hand, the loading capacity (13%) of propo-
fol into the present fatty acid grafted amphiphile [NP(MPEG750)
(Tyr-Lau)Et]3 was higher compared with CP750, and furthermore,
clear and stable 1% propofol solution was obtained without precip-
itation of propofol at least for a few months. The highly hydropho-
bic core environment composed of the present hydrophobic group
Tyr-Lau (logP = 5.26) seems to be more favorable to propofol
compared to docetaxel (logP = 2.59), while the polar micelle core
composed of the polar hexapeptide with less hydrophobicity
(logP = 1.11) favors docetaxel to propofol. We believe that the
present fatty acid grafted amphiphiles are useful for micelle formu-
lation particularly of any highly hydrophobic drugs.

P. G. Avaji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1763–1767
1767
Table 2
Characteristic properties of amphiphilic cyclotriphosphazenes bearing equimolar hydrophilic MPEG and hydrophobic fatty acid
a
Trimer No.
Compounds
Mol. Wt.
logPsum
LCST (°C)b
DLSc (nm)
CMCd (mg/L)
[H2O]
[PBS]
1
2
3
4
5
6
7
[NP(MPEG350)(Tyr-Lau)]3
[NP(MPEG550)(Tyr-Lau)]3
[NP(MPEG550)(Tyr-Myr)]3
[NP(MPEG750)(Tyr-Lau)]3
[NP(MPEG750)(Tyr-Myr)]3
[NP(MPEG1000)(Tyr-Pal)]3
[NP(MPEG1000)(Tyr-Ste)]3
2323.3
2983.7
3067.8
3512.0
3596.1
4472.7
4556.8
3.84
3.02
3.93
2.46
3.37
3.00
3.84
39.4
72.6
69.5
81.4
74.7
82.2
77.3
37.2
65.1
64.3
76.2
71.5
76.6
71.7
5.47
8.02
8.91
8.58
9.21
9.42
10.3
2.95
6.77
4.73
7.80
5.51
4.33
3.32
a
b
c
logPsum = logPTyr-FA + logPMPEG
.
The lower critical solution temperature was identified as the temperature at which the polymer solution (5.0 wt %) became turbid.
The mean diameter of the particles measured for 0.5% solutions at 25 °C by dynamic light scattering method.
The critical micelle concentration was measured by the pyrene method18 at ambient temperature.
d
Supplementary data
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.052.
CMC=2.95mg/L
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Figure 4. The critical micelle concentration (CMC) of cyclotriphosphazene
1
measured by fluorescence probe technique using pyrene.18
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15. The TEM image of [NP(MPEG1000)(Tyr-Ste)]3 deposited on 200 mesh carbon-
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JEM-2100F.
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In conclusion, it is very important to design or apply polymeric
micelles for clinical applications based on the intermolecular inter-
actions between the target drug molecules and the chemical envi-
ronment of the micelle core composed of the hydrophobic
segments of the polymer amphiphiles.
Acknowledgments
This study was supported by a Grant of the Korea Health Tech-
nology R&D Project, Ministry of Health & Welfare, Republic of Kor-
ea (A110562), by National Research Foundation of Korea (NRF)
Grant funded by the Korean Government (MEST) (2012-
0005453), and by the RP-Grant 2012 and Ewha Global Top 5 Grant
2012 of Ewha Womans University.