Supplementary Information
Methods:
1) Molecular Modeling using Molecular Operating Environment (MOE):
Molecular modeling of the different RGD ligands was performed in Molecular Operating
Environment (MOE) software to study the binding interactions. The ligand structure was
constructed in MOE 2011.10 using Molecule Builder tool. The structure was energy
minimized using MMFF94x force field. The structure of integrin complexed with cyclic
RGD is downloaded from the protein databank with PDB id 1L5G. The protein structure
was protonated. Using atom selector, the protein, ligand and pocket was assigned for the
docking study. The proximity in the atoms of co-crystallized ligand, cyclic RGD, was
selected in order to assign the binding pocket. The structure was docked using the Dock
application in MOE. The lowest energy conformation was selected and binding
interactions with the receptor were studied.
2) Synthesis of RGD amphiphiles
2.1 Synthesis of linear RGD amphiphile (C18-ADA5-RGD)
The linear RGD amphiphile was synthesized using standard Fmoc/tBu chemistry. Wang
resin preloaded with aspartic acid (Fmoc-Asp-WR) After swelling the resin in DMF, the
amino group of aspartic acid was deprotected using 20% piperidine in DMF for 30
minutes. After washing the resin with DMF (3 times) and with DCM (3 times), the FmocGly-OH was conjugated in presence of 2 equivalents each of diisopropylcarbodiimide
(DIC) and hydroxybenzotriazole (HOBT) for 2 hours. The washing and deprotection
steps were repeated before conjugation of the next amino acid, Fmoc-Arg(PBf)OH using
the same protocol. Next, the Fmoc-ADA-OH groups were conjugated using 2 equivalents
of
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium
hexafluorophosphate
(HATU), 2 equivalents of HOBT and 4 equivalents of N,N-diisopropylethylamine
(DIPEA) for 3 hours. Once again, the Fmoc group was cleaved using 20% piperidine in
DMF, followed by washing and conjugation of fatty acid was performed by adding 2
equivalents
of
stearic
acid,
2
equivalents
of
PyBOP
(benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate) and 4 equivalents of DIPEA in
mixture of DMF and DCM and treatment for 3 hours. The resin was washed and the
amphiphile was cleaved from the resin by treating with a mixture of TFA:TIS: H2O
(95:2.5:2.5) for 3 hrs. After removal of TFA, the amphiphiles was further precipitated
using ether or ether:hexane (50:50) mixture. The solid precipitate of the amphiphile was
separated by centrifugation and freeze dried before further analysis. The amphiphile was
purified by RP-HPLC for purity >90%.
2.2 Synthesis of cyclic RGD amphiphile (C18-ADA5-c(RGDfK))
Several protocols for synthesis of the cyclic RGD peptide have been reported and
modified for improved yields. The protocol for complete on-resin synthesis of the cyclic
RGD was modified from the protocol by McCusker et al. The O-allyl protected aspartic
acid, Fmoc-Asp-OAll (2.5 equivalents) was loaded on the 2-chloro trityl chloride resin in
the presence of 10 equivalents of N,N-diisopropylethylamine (DIPEA) for 5 hours. After
washing the resin with DMF (3 times) and DCM (3 times), the amino group was
deprotected by treatment with 20% piperidine in DMF for 30 minutes and the wash steps
were repeated. The Fmoc-Gly-OH was added in the presence of 2 equivalents of HOBT
(hydroxybenzotriazole), 2 equivalents of HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3tetramethyl uronium hexafluorophosphate) and 4 equivalents of DIPEA. The subsequent
amino acids in the order of Fmoc-Arg(Pbf)OH, Fmoc-Lys-Dde and Fmoc-Phe-OH were
conjugated using the same deprotection and conjugation protocol. Each conjugation step
was performed for 2 hours. Allyl deprotection was performed using chloroform and Nmethylmorpholine in the presence of palladium catalyst in a N2 atmosphere for 4 hours
followed by amino group deprotection using 20% piperidine in DMF. Cyclization was
carried
out
overnight
in
the
presence
of
PyBOP
(benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate), DIPEA and DMF. This step was
repeated
for
an
additional
dioxocyclohexylidene) ethyl
6
hours.
Further,
the
1-(4,4-Dimethyl-2,6-
(Dde) group was specifically cleaved by using 2%
hydrazine hydrate in DMF. The 8-amino-3,6 dioxaoctanoic acid (ADA) groups were
conjugated to the lysine side chain using the same protocol as for the amino acids.
Finally, stearic acid was conjugated in the presence of PyBOP (4 equivalents) and DIPEA
(8 equivalents). The amphiphile was cleaved from the resin by treating with TFA:TIS:
H2O (95:2.5:2.5) for 3 hrs. After removal of TFA, the amphiphile was precipitated using
ether:hexane (50:50) mixture. The amphiphile was then separated by centrifugation and
freeze dried before further analysis. The amphiphile was purified by RP-HPLC for purity
>90%.
Supplemental Table 1: Binding characteristics of different RGD ligands with αvβ3
integrin obtained from molecular modeling using MOE
Properties
cRGDfK
RGD
Binding Energy Score
-21.8
-5.8
Total Number of Interactions
21
15
FmocNH
O
OtBut
O
O
O
1) 20% Piperidine in DMF
2) Fmoc-Gly-OH, HOBT, DIC
3) 20% Piperidine in DMF
O
NH2
HN
O
OtBut
O
O
O
1) 20% Piperidine in DMF
2) Fmoc-Arg(Pbf)OH, HOBT, DIC
3) 20% Piperidine in DMF
O
O
NH
HN
H2N
NH
NH
O
OtBut
O
O
1) 20% Piperidine in DMF
2) Fmoc-8-amino-3,6-dioxaoctanoic
acid, HATU, HOBT, DIPEA
3) 20% Piperidine in DMF
O
NH(Pbf)
O
Repeat n times
OtBut
O
NH
NH
O
NH
O
O
O
NH2
n
O
O
NH
O
HN
NH(Pbf)
1) 20% Piperidine in DMF
2) Stearic acid, PyBOP, DIPEA
O
OtBut
O
NH
O
NH
O
NH
O
O
O
O
NH
CH3
n
O
NH
O
HN
NH(Pbf)
TFA:TIS:Water 95:2.5:2.5
O
OH
O
NH
O
NH
O
Supplemental
Fig
1: Synthesis nscheme of C18-ADA5-RGD (n=5)
O
O
NH
O
O
NH
HN
NH2
NH
CH3
O
O
NHFmoc
AllO
O
OAll
O
Cl
Cl
O
1) 20% Piperidine in DMF
2) Fmoc-Arg(Pbf)-OH,
HATU, HOBT, DIPEA
Cl
OAll
O
Cl
O
O
OAll
NH
O
Cl
1) 20% Piperidine in DMF
2) Fmoc-Gly-OH, HATU,
HOBT, DIPEA
Fmoc-Asp-OAll, DIPEA
O
NH
O
O
Cl
NHFmoc
O
O
O
O
AllO
NH
NH
HN
NH
O
NH
O
NH
NH(Pbf )
NHFmoc
O
HN
O
NH
HN
1) 20% Piperidine in DMF
2) Fmoc-Phe-OH, HATU, HN
HOBT, DIPEA
O
NH(Pbf )
HN
HN
O
NH
NH
O
NH(Pbf )
O
Cl
NHFmoc
NH(Dde)
O
1) 20% Piperidine in DMF
2) Fmoc-Lys-(Dde)-OH,
HATU, HOBT, DIPEA
NHFmoc
NH(Dde)
CHCl3, N-methyl
morpholine, Pd(PPh3)4 in
inert atmosphere
HO
HO
O
O
Cl
Cl
O
O
O
O
NH
O
O
NH
HN
O
PyBOP, DIPEA in DMF,
2X treatment (16 hours)
NH
O
HN
NH(Pbf )
HN
O
NHFmoc
O
NH(Dde)
HN
O
NH
20% Piperidine in DMF
NH
HN
Cl
HN
O
HN
O
NH
O
NH
NH
NH
O
NH(Pbf )
HN
O
NH(Dde)
O
O
O
N
H
O
H2N
NH(Dde)
Supplemental Fig 2: Scheme for on-resin synthesis of cRGDfK
NH
NH(Pbf )
O
O
Cl
Cl
O
O
O
H
N
O
H
N
O
NH
O
NH
O
O
O
HN
O
HN
2% hydrazine hydrate in DMF
NH
NH
O
O
NH
NH
O
O
NH
NH
NH
NH
NH(Pbf)
NH(Pbf)
NH2
NH(Dde)
Repeat n times
1) Fmoc-8-amino-3,6-dioxaoctanoic acid,
HATU, HOBT, DIPEA
2) 20% Piperidine in DMF
NH(Pbf)
HN
NH
O
O
NH
NH
NH
O
O
NH
O
NH2
n
O
O
NH
O
NH
O
O
O
Cl
Stearic acid, PyBOP, DIPEA
NH(Pbf)
HN
NH
O
O
NH
O
NH
NH
O
O
NH
NH
CH 3
n
O
O
O
NH
O
NH
O
O
O
Cl
TFA:TIS:Water 95:2.5:2.5
NH(Pbf)
HN
NH
O
NH
O
O
NH
NH
O
O
NH
O
O
NH
CH 3
n
O
NH
O
O
HO
NH
O
Supplemental Fig 3: Scheme for on-resin synthesis of C18-ADA5-cRGDfK amphiphile
(n=5)
P
eptidea4
D
ata:P
eptidea40001.D
96A
ug20129:06C
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efl_Franz1209032A
ug201214:51
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ratosP
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xim
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FRV
2.3.4:M
odeR
eflectron,P
ow
er:88,P
.E
xt.@
1700(bin137)
%
Int. 1471m
V
[sum
=76509m
V
]P
rofiles1-52U
nsm
oothed
1337.9
100
90
80
1339.9
70
60
1165.9
50
1166.9
40
30
20
10
0
600
800
1000
1200
1400
M
ass/C
harge
1600
1800
1[c].D9
2000
Supplemental Fig 4: MALDI spectrum of C18-ADA5-RGD amphiphile showing
molecular ion peak
n
z
1
2
0
9
0
3
1
7
M
a
y
2
0
1
3
1
5
:3
4
8
5
,P
.E
x
t.@
2
0
6
7
(b
in
1
5
1
)
1[c].K12
U
n
s
m
o
o
th
e
d
1
5
9
4
.9
1
5
9
6
.9
1
5
0
0
2
0
0
0
M
a
s
s
/C
h
a
rg
e
2
5
0
0
1[c].K12
3
0
0
0
Supplemental Fig 5: MALDI spectrum of C18-ADA5-cRGDfK amphiphile showing
molecular ion peak
1
2
0
9
0
3
2
4
J
a
n
2
0
1
3
1
5
:2
6
6
9
,P
.E
x
t.@
1
1
8
1
(
b
in
1
1
4
)
1[c].C4
U
n
s
m
o
o
th
e
d
1
2
0
3
.0
1
2
0
4
.0
1
2
1
9
.0
1
2
0
0
1
4
0
0
M
a
s
s
/C
h
a
r
g
e
1
6
0
0
1
8
0
0
1[c].C4
2
0
0
0
Supplemental Fig 6: MALDI spectrum of C18-ADA5-GGG amphiphile (sodium adduct
peak at m/z of 1203)
0.85
0.8
0.75
0.7
I3/I1
0.65
0.6
0.55
0.5
-3
-2
-1
0
1
log concentration (μM)
2
3
Supplemental Fig 7: CMC plot of I3/I1 ratio of pyrene v/s concentration of C18-ADA5RGD amphiphile
0.7
0.68
0.66
0.64
I3/I1
0.62
0.6
0.58
0.56
0.54
0.52
0.5
-3
-2
-1
0
1
log concentration of amphiphile (μM)
2
3
Supplemental Fig 8: CMC plot of I3/I1 ratio of pyrene v/s concentration of C18-ADA5cRGDfK amphiphile
0.8
0.75
0.7
I3/I1
0.65
0.6
0.55
0.5
-3
-2
-1
0
log concentration of amphiphile (μM)
1
2
Supplemental Fig 9: CMC plot of I3/I1 ratio of pyrene v/s concentration of C18-ADA5GGG amphiphile
1.2
1
0.8
C18-ADA5-GGG
ln (I0/I) 0.6
C18-ADA5-RGD
C18-ADA5-cRGDfK
0.4
0.2
0
0
1
2
3
[Q] micromolar
Supplemental Fig 10: Plot of ln(I0/I) for pyrene v/s increasing concentration of the
quencher for determination of aggregation number of different amphiphiles
Supplemental Table 2: Difference in tumor inhibition with respect to control at study
endpoint
Treatment
% Tumor Inhibition with
respect to control at End
Point of the Study
Taxol 50 μg/kg twice weekly
28.2
PTX-RGD micelles 50 μg/kg twice weekly
61.8
Taxol 50 μg/kg every alternate day
51.4
PTX-RGD micelles 50 μg/kg alternate day
68.9