Salts responsive nanovesicles through π-Stacking induced self-assembly of backbone modified tripeptides

Article abstract of DOI:10.1166/jnn.2011.4219

A set of backbone modified peptides of general formula Boc-Xx-m-ABA-Yy-OMe where m-ABA is meta-aminobenzoic acid and Xx and Yy are natural amino acids such as Phe, Gly, Pro, Leu, Ile, Tyr and Trp etc., are found to self-assemble into soft nanovesicular st

Full text of DOI:10.1166/jnn.2011.4219

Copyright © 2011 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 11, 6747–6756, 2011
Salts Responsive Nanovesicles Through ꢀ-Stacking
Induced Self-Assembly of Backbone Modified Tripeptides
Pradyot Koley1, Michael G. B. Drew2, and Animesh Pramanik1ꢀ∗
1Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700009, India
2School of Chemistry, The University of Reading, Whiteknights, Reading RG6 6AD, UK
A set of backbone modified peptides of general formula Boc-Xx-m-ABA-Yy-OMe where m-ABA is
meta-aminobenzoic acid and Xx and Yy are natural amino acids such as Phe, Gly, Pro, Leu, Ile, Tyr
and Trp etc., are found to self-assemble into soft nanovesicular structures in methanol-water solution
(9:1 by v/v). At higher concentration the peptides generate larger vesicles which are formed through
fusion of smaller vesicles. The formation of vesicles has been facilitated through the participation of
various noncovalent interactions such as aromatic ꢀ-stacking, hydrogen bonding and hydrophobic
interactions. Model study indicates that the ꢀ-stacking induced self-assembly, mediated by m-ABA
is essential for well structured vesicles formation. The presence of conformationally rigid m-ABA in
the backbone of the peptides also helps to form vesicular structures by restricting the conformational
entropy. The vesicular structures get disrupted in presence of various salts such as KCl, CaCl2,
N(n-Bu)4Br and (NH4ꢁ2SO4 in methanol-water solution. Fluorescence microscopy and UV studies
reveal that the soft nanovesicles encapsulate organic dye molecules such as Rhodamine B and
Acridine Orange which could be released through salts induced disruption of vesicles.
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Keywords: Nanovesicles, Peptides, Self-Assembly, Responsive.
IP: 66.109.184.11 On: Fri, 30 Oct 2015 10:26:45
Copyright: American Scientific Publishers
1. INTRODUCTION
noncovalent interactions are complementary and effective
in organic solvents, it opens a way to fabricate nano-
structures in a more wider range of solvent systems.
Several bioinspired designs of soft assemblies based on
aromatic ꢀ–ꢀ interactions have been reported in recent
times.34–39 In this paper we are interested in developing
simple molecular systems which will produce responsive
supramolecular assemblies through hydrogen bonding and
aromatic ꢀ-stacking induced self-assembly.
Keeping this in view several backbone modified
tripeptides of general formula Boc-Xx-m-ABA-Leu-OMe
(m-ABA: meta-aminobenzoic acid) where Xx is Phe in
peptide I, Gly in peptide II, Pro in peptide III, Ile in
peptide IV and Tyr in peptide V have been designed
by incorporating m-aminobenzoic acid at the centre to
invoke aromatic ꢀ–ꢀ interactions in the self-assembly
(Fig. 1). At the same time the conformationally rigid
m-ABA in the backbones of the peptides will help to pro-
duce supramolecular assemblies by restricting the confor-
mational entropy.18ꢁ40–45 Moreover the presence of m-ABA
may provide proteolytic stability to the self-assembled
supramolecular structures.18 Peptides containing Leu at
position 1 such as Boc-Leu-m-ABA-Yy-OMe, where Yy
is Ile in peptide VI and Trp in peptide VII have also been
designed to examine whether the self-assembly of all these
Design and construction of soft molecular assemblies that
can respond to external stimulus are becoming increas-
ingly important because of their diverse applications in
drug delivery, carrying biologically relevant molecules and
sensing various biochemical processes.1–3 External stim-
uli such as temperature, pH, light, electric field, chem-
ical and ionic strength may trigger disruption of soft
assemblies by modifying shape and surface properties.4–12
It has been observed that peptides are efficient building
blocks for fabricating nanoscopic soft assemblies respon-
sive to various external stimuli.4–17 Generally amphiphilic
small peptides self-assemble into nanovesicles in aqueous
medium through interactions occurring between hydropho-
bic and hydrophilic parts.18–21 Surfactant-like peptides also
undergo self-assembly to form nanovesicles in polar sol-
vent due to hydrophobic and hydrophilic interactions.22–25
However, recent studies have demonstrated that the for-
mation of nanostructures could also be facilitated through
the participation of other non-covalent interactions such as
hydrogen bonding and aromatic ꢀ-stacking, with or with-
out the presence of hydrophobic effect.26–33 Since these
∗Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2011, Vol. 11, No. 8
1533-4880/2011/11/6747/010
doi:10.1166/jnn.2011.4219
6747

Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
Koley et al.
(a) Boc-Phe-m-ABA-OMe (1). Initially N-terminal
protection with Boc group of L-Phenyl alanine was
performed using the literature method.47 The fragment
Boc-Phe-OH (1.5g, 5.66 mmol) was dissolved in a
mixture of dichloromethane (DCM, 6 ml) and dimethyl-
formamide (DMF, 5 ml). H-mABA-OMe obtained from
its hydrochloride (2.12 g, 11.31 mmol) was then added
followed by addition of DCC (1.75 g, 8.49 mmol) and
HOBT (0.76 g, 5.66 mmol) in ice-cold conditions. The
reaction mixture was stirred at room temperature for
1 day. The precipitated N,Nꢀ-dicyclohexylurea (DCU) was
filtered off. The organic layer was diluted with 30 ml
ethyl acetate and washed with 1N HCl (3×30 ml), brine,
1M Na2CO3 solution (3 × 30 ml) and then again with
brine. The solvent was dried over anhydrous Na2SO4 and
evaporated in vacuo to give a waxy colorless solid. Yield:
(2.0 g, 88.89%).
O
O
H
N
H
N
O
N
H
OMe
O
O
R1
H
H
R2
Peptide I: R1= -CH2-C6H5,
Peptide II: R1= -H,
IV: R1= -CH(CH3)-CH2-CH3, R2= -CH2-CH-(CH3)2
Peptide V: R1= -CH2-(C4H4)-p-OH, R2= -CH2-CH-(CH3)2
VI: R1= -CH2-CH-(CH3)2,
Peptide VII: R1= -CH2-CH-(CH3)2
R2= -CH2-CH-(CH3)2
R2= -CH2-CH-(CH3)2
Peptide
R2= -CH(CH3)-CH2-CH3
R2 = -CH2-(3-indonyl)
Peptide
O
N
O
H
N
OMe
H
N
O
H
O
O
Peptide III
(b) Boc-Phe-m-ABA-OH (2). 1 (2.0 g, 5.02 mmol) was
dissolved in methanol (20 ml) and 2N NaOH (10 ml)
was added. The reaction mixture was stirred for 1 day at
room temperature. The progress of the reaction was mon-
itored by TLC. After completion of reaction the methanol
was evaporated. The residue was diluted with water and
washed with diethyl ether. The aqueous layer was cooled
in an ice-bath and then neutralized by using 2N HCl and
extracted with ethyl acetate. The solvent was evaporated
in vacuo to give a waxy colorless solid. Yield: (1.75 g,
Fig. 1. Schematic representation of peptides I–VII.
peptides I–VII is sequence specific or not (Fig. 1). Model
study has been carried out with peptides Boc-Leu-Phe-
Ile-OMe (VIII) and Boc-Leu-Ile-Val-OMe (IX), where
the centrally placed m-ABA has been replaced by natural
amino acids to assess the role of m-ABA in the formation
of supramolecular assemblies. The dye entrapment prop-
erty and responsive behavior of the supramolecular assem-
blies to external stimuli have been explored.
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90.67%).
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(c) Boc-Phe-m-ABA-Leu-OMe (peptide I). 2 (1.75 g,
Copyright: American Scientific Publishers
4.56 mmol) was dissolved in DMF (10 ml). Leu-OMe
2. EXPERIMENTAL DETAILS
obtained from its hydrochloride (1.65 g, 9.09 mmol) was
added, followed by addition of DCC (1.41 g, 6.84 mmol)
and HOBT (0.62 g, 4.58 mmol) in ice-cold conditions.
The reaction mixture was stirred at room temperature
for 2 days. The precipitated DCU was filtered off. The
organic layer was diluted with 30 ml ethyl acetate and
washed with 1N HCl (3×30 ml), brine, 1M Na2CO3 solu-
tion (3 × 30 ml) and then again with brine. The solvent
was then dried over anhydrous Na2SO4 and evaporated
in vacuo, to give a colorless solid compound. Purification
was done using silica gel as stationary phase and ethyl
acetate-petroleum ether mixture as eluent. Single crystals
were grown from CHCl3-petroleum ether mixture by slow
evaporation.
2.1. General Methods and Materials
All amino acids, meta-amino benzoic acid, HOBt
(1-hydroxybenzotriazole) and DCC (dicyclohexylcarbodi-
imide) were purchased from Sigma chemicals.
2.2. Synthesis of the Peptides
The peptides were synthesized by conventional solu-
tion phase methodology.46 The t-butyloxycarbonyl (Boc)
group was used for N-terminal protection and the
C-terminus was protected as the methyl ester. Cou-
plings were mediated by N,Nꢀ-dicyclohexylcarbodiimide
(DCC)/1-hydroxybenzotriazol (HOBT). Deprotection of
the methyl ester was performed using the saponifica-
tion method. All intermediates were characterized by thin
layer chromatography on silica gel and used without fur-
ther purification. Final peptides were purified by column
chromatography using silica gel (100–200 mesh) as the
stationary phase. Ethyl acetate and petroleum ether mix-
ture was used as the eluent. The reported peptides I–IX
Yield: (2.1 g, 90.13%). Mp = 120–122 ꢁC (from CHCl3-
petroleum ether); IR ꢂmax(KBr)/cm−1 : 3297, 3063, 2958,
1749, 1674, 1548, 1445; 1H NMR (300 MHz, CDCl3,
ꢁ
25 C, TMS) ꢃ ppm: 9.27 (1 H, s, m-ABA (2) NH); 7.92
(1 H, d, J = 7.8 Hz, m-ABA (2) Hd); 7.75 (1 H, s, m-ABA
(2) Ha); 7.16–7.36 (7 H, m, m-ABA (2) Hb, Hc and Phe
(1) phenyl ring protons); 6.83 (1 H, br, Leu (3) NH); 5.09
(1 H, d, J = 7.5 Hz, Phe (1) NH); 4.67–4.74 (2 H, m,
Leu (3) & Phe (1) CꢄHs); 3.81 (3 H, s, –OCH3ꢅ; 3.04–
3.21 (2 H, m, Phe (1) CꢆHs); 1.63–1.67 (2 H, m, Leu (3)
CꢆHs); 1.38 (9 H, s, Boc CH3Sꢅ; 0.83-0.97 (7 H, m, Leu
(3) CꢇH and CꢃHs).
1
were characterized by H NMR spectroscopy, 13C NMR
spectroscopy, microanalysis and IR spectroscopy while
the structures of peptides I–III were confirmed by X-ray
crystallography.
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Koley et al.
Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
13C NMR (75 MHz, CDCl3ꢅꢃ ppm: 173.87, 171.35,
167.55, 156.69, 136.98, 135.73, 134.73, 129.38, 129.20
(2C), 128.78 (2C), 127.25, 124.08, 123.05, 117.31, 81.07,
56.21, 52.15, 51.50, 40.49, 38.54, 28.30 (3C), 24.77,
23.29, 21.19.
transferred onto a freshly cleaved mica surface and the
sample coated mica was dried for 12 hours at room tem-
perature. With these samples coated mica surfaces, AFM
studies were carried out using a VEECO, Multimode,
Nanoscope-IIIA atomic force microscope operating under
Tapping mode (RTESP Tip). The force constant was 20–
80 N m−1, while the resonant frequency was 286.17 kHz.
Anal. Calcd for C28H37N3O6: C, 65.73; H, 7.29; N, 8.21.
Found: C, 65.66; H, 7.22; N, 8.17%.
Synthesis and characterization of peptides II–IX were
carried out using the same procedure.
2.3.4. Dynamic Light Scattering (DLS) Measurements
Freshly prepared solutions of peptides I–III (6.0 mM)
in methanol-water (9:1 by v/v) were used for dynamic
light scattering measurements using a Nano-S-1600,
MALVERN, USA instrument, after ultrasonication of the
solutions for 30 min.
2.3. Characterization Techniques
2.3.1. Scanning Electron Microscopy (SEM).
SEM and FE-SEM imaging were carried out to investigate
the morphology of the nanostructures. In general, freshly
prepared solutions of peptides (10.0 mM) in methanol-
water (9:1 by v/v) were taken on glass cover slips and
evaporated to dryness for 24 hours. A gold coating was
applied to the top of the samples to make it conductive
for analysis. Then, SEM measurements were performed
on a S3400N, HITACHI, JAPAN instrument and JEOL
JSM-6700F instrument at 5.0 kV to 10.0 kV voltage and
5000 × to 40 000 × magnification. For salts triggered dis-
ruption studies 10.0 mM solutions of various salts such
as KCl, CaCl2, N(n-Bu)4Br and (NH4ꢅ2SO4 in methanol-
water (1:1 by v/v) were added into the freshly prepared
2.3.5. Thermogravimetric Analysis (TGA)
A solution of peptide I (6.5 mg mL−1ꢅ in methanol-water
(9:1 by v/v) was aged for 7 days in open air at r. t. and then
dried under vacuum. This sample was analyzed on a Met-
tler Toledo TGA/SDTA 851 thermal analyzer in a dynamic
atmosphere of dinitrogen (flow rate = 30 cm3 min−1ꢅ. The
saꢁmple was heated in an alumina crucible at a rate of
5 C min−1
.
2.3.6. Fluorescence Microscopy and UV Studies
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10.0 mM methanol-water solution of peptide I in 1:1 (v/v)
proportion and incubated for 12 hours and followed by
scanning electron microscopic imaging.
A mixture of solution of peptide I in methanol-water
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(9:1 by v/v) and Rhodamine B in a 1:1 v/v ratio (final
concentration of each of the substance being 10−4 M)
was incubated for 1 day at r. t. After 1 day one drop
(20 ꢈL) of the solution was loaded on the glass slide and
dried at r. t. under vacuum. The excess dye was removed
from glass slide by repeated washing with deionized water.
Then the dye loaded glass slide was dried at r. t. under
vacuum. Finally these dye entrapped vesicular structures
were examined under a fluorescence microscope (Fluores-
cence Microscope Olympus BX 61). Similar experiment
was done with the dye Acridine Orange also. For salt
triggered disruption studies, solutions of various salts in
methanol-water (9:1 by v/v) were added on the 1 day aged
solution of peptide I and Rhodamine B (final concentra-
tion of each of the substance being ∼10−4 M) and the
resultant solution was incubated for 12 hours. After that
one drop (20 ꢈL) of the solution was loaded on the glass
slide and dried at r. t. under vacuum. Finally the ruptured
vesicles and release of dyes were examined on the same
fluorescence microscope.
Copyright: American Scientific Publishers
2.3.2. High Resolution Transmission Electron
Microscopy (HR-TEM)
High resolution transmission electron microscopy (HR-
TEM) was also carried out to investigate the morphology
of nanostructures. Freshly prepared solutions of peptides
(6.0 mM) in methanol-water (9:1 by v/v) were sonicated
with UC 250W, INECO ultrasonicator for 30 mins, and
one drop of each of these solutions was taken in a carbon-
coated copper grid (300 mesh) and evaporated to dryness
under vacuum for 12 hours. With these grids TEM studies
were carried out using a JEOL JEM-2100 electron micro-
scope and TECNAI G2 SPIRIT Bio TWIN electron micro-
scope with 80 kV operating voltage. For salts triggered
disruption studies, 6.0 mM solutions of various salts such
as KCl, CaCl2, N(n-Bu)4Br and (NH4ꢅ2SO4 in methanol-
water (1:1 by v/v) were added into the freshly prepared
6.0 mM methanol-water solution of peptide I in 1:1 (v/v)
proportion and incubated for 12 hours followed by trans-
mission electron microscopic imaging.
The Fluorescence emission spectra (730–530 nm) of
peptide I was recorded in methanol-water solution (9:1 by
v/v) with a Fluorescence Spectrometer Perkin Elmer LS
55 using ∼1% Attenuator Assy. The spectrums were taken
using an excitation wavelength of 540 nm. The electronic
absorption spectra (800–200 nm) of the peptides were
recorded in methanol-water solution (9:1 by v/v) with a
Hitachi U-3501 spectrophotometer.
2.3.3. Atomic Force Microscopy (AFM)
One drop (10 ꢈL) of freshly prepared methanol-water
(9:1 by v/v) solutions of peptides I–III (6.0 mM) was
J. Nanosci. Nanotechnol. 11, 6747–6756, 2011
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Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
Koley et al.
(a)
2.3.7. Single Crystal X-ray Diffraction Study
Diffraction data for the three peptides I, II and III, were
obtained with MoKꢄ radiation at 150 K using the Oxford
Diffraction X-Calibur CCD System. The crystals were
positioned at 50 mm from the CCD. 321 frames were mea-
sured with a counting time of 10 s. Data analyses were car-
ried out with the Crysalis program.48 The structures were
solved using direct methods with the Shelxs97 program.49
Non-hydrogen atoms were refined with anisotropic thermal
parameters. The hydrogen atoms bonded to carbon were
included in geometric positions and given thermal parame-
ters equivalent to 1.2, or 1.5 times for methyl groups, times
those of the atom to which they were attached. There was
disorder in parts of the side groups in peptide I which also
contained two water molecules, refined with 50% occu-
pancy. The structures were refined on F2 using Shelxl97.49
The CIF files of the 3 structures have been deposited at
the Cambridge Crystallographic Data Centre with refer-
ence numbers CCDC 767778 to 767780 inclusive.
(b)
a
c
b
Fig. 2. (a) The molecular duplex of peptide II in the solid state formed
through aromatic ꢀ–ꢀ interactions and hydrogen bonds, shown as dotted
lines; (b) Packing pattern of peptide II showing the formation ꢆ-sheet
layer in the ab plane.
3. RESULTS AND DISCUSSION
3.1. Single Crystal X-ray Diffraction and UV Studies
of Self-Assembly
The single crystal X-ray diffraction studies of the crystals
ꢆ-sheet layers along two axes may lead to the formation
of spherical structures under suitable condition.20
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of peptides I–III grown from CHCl3-petroleum ether mix-
IP: 66.109.184.11 On: Fri, 30 Oct 2015 10:26:45
ture revealed that all three structures, though not isomor-
The existence of aromatic ꢀ-stacking interactions in
the solid state was further probed by UV studies. The
highly diluted methanolic solutions (0.015 mM) of the
peptides I–III show absorption maxima around 225 nm
due to aromatic chromophore (Fig. 3), where the pep-
tide molecules are not self-assembled and there are no
ꢀ-stacking interactions. But the solid materials of peptides
I–III grown from methanol-water show absorption max-
ima around 305 nm. The substantial red shift (∼80 nm)
on going from solution to solid state may be attributed to
Copyright: American Scientific Publishers
phous, contained four molecules, named A, B, C, D in the
asymmetric unit. All the molecules adopt similar extended
structures with the centrally placed m-ABA providing an
all trans extended configuration. In the solid state all three
structures form dimers via ꢀ–ꢀ interactions between the
phenyl rings of m-ABA. In peptide I, the two phenyl rings
intersect at angles of 15.7ꢁ in the AB dimer and 16.4ꢁ in
the CD dimer with closest CꢉꢉꢉC contacts of 3.55, 3.48 Å
respectively. In peptide II, angles are 30.8ꢁ and 15.8ꢁ and
shortest distances 3.56, 3.50 Å respectively (Fig. 2(a))
while in peptide III, dimensions are 15.7ꢁ, 16.4ꢁ, 3.55,
3.48 Å respectively. The dimers are further stabilized by
four strong intermolecular hydrogen bonds between Boc-
COꢉꢉꢉH-N-m-ABA and Xx(1)-COꢉꢉꢉH-N-Leu (Fig. 2(a)).
The formation of these molecular dimers AB and CD,
described above, is common to all three peptides but fur-
ther packing of the dimers facilitated by hydrogen bond
formation involving N(3) is dependent upon the nature of
the further residues in the peptides.
0.8
Peptide I
Peptide II
Peptide III
0.6
0.4
0.2
0.0
The dimers of peptides I–III are packed into ꢆ-sheet
layers through intermolecular hydrogen bonds and van
der Waals interactions (Fig. 2(b)). The solid state pack-
ing clearly demonstrates that all the peptides I–III form
layers of ꢆ-sheets through molecular self-assembly stabi-
lized by various noncovalent interactions such as aromatic
ꢀ–ꢀ interactions, hydrogen bonding and van der Waals
interactions. It has been proposed that the closure of the
250
300
350
400
Wavelength (nm)
Fig. 3. UV absorption spectra indicate a significant red shift (∼ 80 nm)
on going from solution (0.015 mM) to vesicular solid state of peptide
I–III due to aromatic ꢀ–ꢀ interactions.
6750
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Koley et al.
Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
the ꢀ-stacking interactions among the aromatic rings in
the fusion of smaller vesicles, which is evident from the
SEM images where fused vesicles are observed (data not
shown). The time-dependent SEM imaging of peptide II
after 6 hrs and 24 hrs clearly shows the intermediate
structures affording vesicle fusion (Fig. 6). TEM images
of peptides I and II at higher concentration (10.0 mM)
also show the formation of larger vesicles (∼400–800 nm)
(Fig. 5(c), representative data shown). Interestingly, some
intermediate states of fused vesicles of peptide II are also
observed in the TEM image (Fig. 5(d)). The AFM studies
of peptides I–III with 6.0 mM solutions in methanol-water
(9:1 by v/v) show the formation of vesicular structures
with an average diameter 20–200 nm (Fig. 7(a), represen-
tative data shown).
the solid state due to self-assembly.50
3.2. SEM, TEM and AFM Studies
The scanning electron micrographs (SEM) reveal that the
peptides I–III can generate spherical objects from CHCl3-
petroleum ether mixture (3:7 by v/v), but the spheres are
found in aggregated form (data not shown). Therefore we
decided to use more polar methanol-water mixture (9:1 by
v/v) for generating isolated spherical objects. The SEM
images show that the peptides I–V generate well structured
spherical objects of an average diameter 0.5–2 ꢈm, when
freshly prepared methanol-water (9:1 by v/v) solutions of
peptides (10.0 mM) were dried on glass surfaces by slow
evaporation (Figs. 4(a–c), representative data shown). Pep-
tides VI and VII where Leu has been placed at position
1 and Ile and Trp at position 3 also show similar sphere
forming properties (data not shown), indicating that there
is no restriction in choosing the first and third residues
of the peptides for these spherical structures formations
as long as they are hydrophobic in nature. Interestingly
the SEM images of the peptides also reveal some cracked
and opened-up hollow spheres which indicate the hollow
nature of the spherical objects (Figs. 4(b and d)). These
porous structures are formed when the trapped volatile sol-
vent such as methanol escapes from the surface during the
3.3. Dynamic Light Scattering (DLS) Studies
To probe the size distributions of nanovesicles in the
solution phase, DLS experiments were carried out with
6.0 mM solutions of peptides I–III in methanol-water
(9:1 by v/v), after ultrasonication of the solutions
for 30 min. Although SEM and TEM studies reveal
the formation of both bigger and smaller nanovesicles
simultaneously, DLS data demonstrate the formation of
monodispersed vesicles with a distribution of hydrody-
namic diameters centered on 85.0 nm in peptide I,
105.7 nm in peptide II and 164.2 nm in peptide III
(Fig. 7(b), representative data shown). Significantly all the
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drying process.51–56 This phenomenon is quite similar to
IP: 66.109.184.11 On: Fri, 30 Oct 2015 10:26:45
that observed when engendering porosity in polymer sam-
peptides I–III generate small sized nanovesicles in the
solution phase compared to vesicles generated on the solid
surfaces.
Copyright: American Scientific Publishers
ples with the help of porogens.57
The SEM studies confirm that the peptides VIII and
IX where the centrally placed m-ABA has been replaced
by natural hydrophobic amino acids can not produce well-
structured spherical objects from methanol-water (9:1 by
v/v) solutions. The result indicates that the ꢀ-stacking
induced self-assembly, mediated by m-ABA is essential for
well structured spherical objects formation. The confor-
mational constrain induced by m-ABA plays an important
role in the formation of supramolecular spherical assem-
blies by restricting the conformational entropy. Although
hydrophobic interaction helps in the self-assembly, this
alone cannot lead to the formation of well-structured
spherical objects as evident from the results of peptides
VIII and IX.
To examine the fusion process of the vesicles in the
solution phase time-dependent DLS measurements were
carried out with a 10.0 mM solution of peptide II in
methanol-water (9:1 by v/v). The distributions of hydro-
dynamic diameters with the increase in time are plotted
in Figure 8(a). The plot clearly shows that the hydro-
dynamic diameter of the vesicles increases steadily upto
24 hrs after that the fusion of vesicles stops as there is no
further significant change in diameter. The plot of hydro-
dynamic diameters of vesicles against time shows a con-
sistent increase in the size of the vesicles upto 24 hrs
(Fig. 8(b)).
The TEM studies reveal that the peptides I–III form
vesicular structures from 6.0 mM solutions of peptides
in methanol-water (9:1 by v/v) with an average diameter
20–200 nm (Figs. 5(a and b), representative data shown),
which is much less than the average diameter observed
in SEM studies (Fig. 4). TEM images clearly indicate the
layer-like boundary and hollow nature of self-assembled
structures of the peptides (Fig. 5). Therefore the spheri-
cal objects observed in the SEM images are actually vesi-
cles. The results indicate that the size of the vesicles is
dependent on concentration of the peptides. At higher con-
centration (10.0 mM) larger vesicles are formed through
3.4. Thermo Gravimetric (TGA) and Differential
Thermal Analysis (DTA)
The TGA thermogram of representative peptide
I
nanovesicles shows ∼21% weight loss between room tem-
ꢁ
perature and ∼240 C (Fig. 9). This may be attributed to
the loss of methanol and water from the confines of peptide
vesicular structures. The result ꢁindicates that the vesicular
structures are stable up to 240 C, after which the peptide
decomposes. The DTA plot shows that the loss of solvent
molecules from the vesicular structures is an endothermic
process.
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Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
Koley et al.
Fig. 4. SEM images of nanovesicles of (a) peptide I, (b) peptide IV and (c) peptide V, generated from methanol-water (9:1 by v/v) solution (10.0 mM);
(d) Showing the hollow sphere of peptide IV.
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Fig. 5. HR-TEM images of nanovesicles of (a) peptide I and (b) peptide II generated from 6.0 mM methanol-water (9:1 by v/v) solutions of peptides;
(c) TEM image of bigger nanovesicles of peptide II at higher magnification generated from 10.0 mM solution; (d) TEM image of fused vesicles of
peptide II.
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Koley et al.
Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
Fig. 6. Time-dependent SEM images of peptide II (a) freshly prepare solution, (b) after 6 hrs and (c) after 24 hrs, showing the intermediate structures
affording fusion.
3.5. Formation and Disruption of Vesicles
nanovesicles may be envisaged by considering the closure
of the ꢆ-sheet-like layers in two different directions simul-
taneously as illustrated in Figure 10.20ꢁ21 At higher con-
centration (10.0 mM), the peptides form bigger vesicles
which are formed through the fusion of small vesicles. The
driving forces behind the fusion process may be the release
of strain in the initially formed vesicles, which have high
curvature. By fusion into larger vesicles, curvature energy
decreases, thus leading to a thermodynamically more sta-
ble state.59
It is evident from the TEM, SEM, and AFM images
that the hydrophobic peptides I–VII with centrally placed
m-ABA generate vesicular structures from polar methanol-
water mixture. The m-ABA mediated ꢀ-stacking inter-
actions play an important role in the self-assembly.
Interestingly, the role of aromatic ꢀ-stacking interactions
in providing energetic contributions as well as direction-
ality has been proposed for self-assembled protein/peptide
aggregates.58 Crystal packing shows that the peptides
I–III form ꢆ-sheet layers through molecular self-assembly
(Fig. 2(b), representative data shown). The formation of
(a)
Time
30 Min
2.5 Hrs
4.5 Hrs
6.5 Hrs
24 Hrs
26 Hrs
29 Hrs
48 Hrs
53 Hrs
5 Days
50
(a)
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30
1.00
20
10
0.50
100
200
300
400
500
600
0
Hydrodynamic diameter (nm)
0
0.50
1.50
1.50
(b)
µm
0.0 nm
5.0 nm 10.0 nm
300
250
200
150
100
50
50
40
30
20
10
0
(b)
0
10
20
30
40
50
60
Time (h)
0.1
1
10
100
1000
10000
Fig. 8. (a) Plot showing the distributions of hydrodynamic diameters
of the nanovesicles of peptide II in 10.0 mM methanol-water (9:1 by
v/v) solution at different time in DLS study; (b) Plot showing the vari-
ation of hydrodynamic diameter of the vesicles of peptide II with time.
Hydrodynamic diameters of highest intensities were plotted against time.
Diameter (nm)
Fig. 7. (a) AFM topography image and (b) distribution diameter of
nanovesicles of peptides I in DLS study of 6.0 mM methanol-water (9:1
by v/v) solution of peptide.
J. Nanosci. Nanotechnol. 11, 6747–6756, 2011
6753

Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
Koley et al.
°C
0
mg
6.5
for 12 hours and followed by SEM and TEM imag-
ing. In all cases disruption of nanovesicles were observed
(Figs. 11(a and b)). The addition of salts to the solutions
causes the simultaneous breakage of various weak nonco-
valent interactions present in the vesicular structures, such
as aromatic ꢀ-stacking, hydrogen bonding and hydropho-
bic interactions.61 All these effects together lead to the
disruption of nanovesicles.
Peptide I (6.4838 mg)
–1
–2
–3
–4
–5
6.0
5.5
5.0
4.5
4.0
3.5
40
60
80 100 120 140 160 180 200 220 240 260 280 °C
–6
3.6. Encapsulation and Release of Dyes
0
5
10
15
20
25
30
35
40
45
50 min
Entrapment of organic dye molecules such as
Rhodamine B and Acridine Orange within the vesicular
Fig. 9. The black curve indicates TGA and red curve indicates DTA.
structures of peptide I has been examined.21ꢁ34ꢁ35ꢁ62ꢁ63
A
methanol-water (9:1 by v/v) solution of Rhodamine B
was added to a methanol-water (9:1 by v/v) solution of
peptide I so that the final concentration of each substance
was maintained at ∼10−4 M in the resultant solution.
One drop of the one day aged above solution was loaded
on a glass slide and dried at room temperature and then
examined under fluorescence microscope after washing
out the excess dyes with deionized water for several
times. Similar study was carried out with Acridine Orange
also. Visualization of the Rhodamine B and Acridine
Orange entrapped vesicular structures strongly supports
the encapsulation phenomenon (Figs. 11(c and e)). This
phenomenon was further confirmed by fluorescence emis-
Fig. 10. Schematic representation of a possible mode of vesicle forma-
tion from ꢆ-sheet layers of peptides.
In order to understand the driving forces for such self-
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sion study. Initially the fluorescence emission spectra of a
assembly and the softness of the vesicular structures, the
salts triggered disruption studies were carried out.34ꢁ60
In this experiment methanol-water solutions of various
salts such as KCl, CaCl2, N(n-Bu)4Br and (NH4ꢅ2SO4 (6–
10 mM) were added into freshly prepared methanol-water
solutions of peptide I (6–10 mM) separately and incubated
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10−4 M solution of pure Rhodamine B in methanol-water
Copyright: American Scientific Publishers
(9:1 by v/v) solution was recorded, which showed emis-
sion band around 570 nm (Fig. 12(a)). Then fluorescence
emission spectra of a methanol-water (9:1 by v/v) solution
of Rhodamine B and peptide I, where the concentration of
Fig. 11. (a) SEM and (b) TEM images of ruptured vesicles of peptide I after addition of KCl solution and incubation for 12 hours; and
(c) Rhodamine B entrapped nanovesicles of peptide I observed under fluorescence microscope (one day aged solution of dye-loaded vesicles); (d) flo-
rescence microscopic image of ruptured vesicle after addition of KCl solution to the dye loaded vesicular solution of peptide I and incubation for
12 hours. (e) and (f) are the fluorescence images of Acridine Orange dye loaded and ruptured nanovesicles of peptide I respectively.
6754
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Koley et al.
Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
each substance was maintained at ∼10−4 M was recorded.
In first 3 hours no significant change was observed in the
fluorescence intensity compared to the pure Rhodamine B
solution. After 4 hours the fluorescence intensity decreased
steadily over next 8 hours showing encapsulation of dye
molecules by the vesicles (Fig. 12(a)). Further the dye
loaded vesicular structures of peptide I were found to
get disrupted in presence of KCl causing the release of
entrapped Rhodamine B when observed under fluores-
cence microscope (Fig. 11(d)). Similarly the rapture of
Acridine Orange loaded vesicles of peptide I by KCl was
also observed by fluorescence microscopy (Fig. 11(f)).
The encapsulation of Acridine Orange by the vesicles of
peptides I was confirmed by recording UV absorption
spectra, in which a steady decrease of absorption intensity
of Acridine Orange with time was observed upto 12 hours
(Fig. 12(b)). The initial enhancement in the absorbance
after addition of dye may be due to weak interactions
between dye and peptide molecules.
4. CONCLUSION
It has been observed that the backbone modified peptides
of general formula Boc-Xx-m-ABA-Yy-OMe (m-ABA:
meta-aminobenzoic acid) where Xx and Yy are natural
amino acids such as Phe, Gly, Pro, Leu, Ile, Tyr and Trp
etc. can self-assemble into soft nanovesicular structures
in methanol-water solution. The formation of vesicles has
been facilitated through the participation of various nonco-
valent interactions such as aromatic ꢀ-stacking, hydrogen
bonding and hydrophobic interactions. The model study
indicates that the ꢀ-stacking induced self-assembly, medi-
ated by m-ABA is essential for well structured vesicles for-
mation. The conformational constrain induced by m-ABA
plays an important role in the formation of nanovesi-
cles by restricting the conformational entropy. Although
hydrophobic interaction helps in the self-assembly, this
alone cannot lead to the formation of well-structured vesi-
cles as evident from the results of peptides VIII and IX,
where m-ABA is replaced by natural amino acids. The
vesicular structures are responsive to various salts such
as KCl, CaCl2, N(n-Bu)4Br and (NH4ꢅ2SO4and can be
disrupted by the simple addition of them. Fluorescence
microscopy and UV studies reveal that the nanovesicles
can encapsulate dye molecules which could be released by
addition of salts. These soft nanovesicles may find appli-
cations as delivery vehicles for entrapment and transport
(a)
Rhodamine B
250
200
150
100
50
Rhodamine B+Peptide I (0 Hrs)
Rhodamine B+Peptide I (3 Hrs)
Rhodamine B+Peptide I (4 Hrs)
Rhodamine B+Peptide I (6 Hrs)
Rhodamine B+Peptide I (8 Hrs)
Rhodamine B+Peptide I (12 Hrs)
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of natural and unnatural molecules. Currently extensive
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research interest has been directed towards studies of vesi-
Copyright: American Scientific Publishers
cle fusion.64ꢁ65 It has been observed that larger vesicles
are formed through fusion of small vesicles. This may be
utilized for the model study to gain more insights about
the formation, transport, and targeted fusion of endosomal
vesicles in cell-free conditions.
0
560
580
600
620
640
660
Acknowledgments: The financial assistance of UGC,
New Delhi is acknowledged [Major Research Project,
No. 32-190/2006(SR)]. We acknowledge the financial
assistance of Centre for Research in Nanoscience & Nano-
technology (CRNN), University of Calcutta. We thank
Dr. Prabir K. Maiti and Anirban Chakraborty of C. U. for
providing SEM (TEQIP) and AFM (TEQIP) instrumen-
tal facilities. Pradyot Koley would like to thank CRNN
for a senior research fellowship (SRF). We thank EPSRC
and the University of Reading, UK for funds for Oxford
Diffraction X-Calibur CCD diffractometer.
Wavelength (nm)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(b)
Acridine Orange (AO)
AO+Peptide I (15 Mins)
AO+Peptide I (1 Hrs)
AO+Peptide I (6 Hrs)
AO+Peptide I (12 Hrs)
References and Notes
1. C. de las, H. Alarcón, S. Pennadam, and C. Alexander, Chem. Soc.
Rev. 34, 276 (2005).
200
250
300
350
400
450
550
550
2. R. Yerushalmi, A. Scherz, M. E. van der Boom, and H.-B. Kraatz,
J. Mater. Chem. 15, 4480 (2005).
Wavelength (nm)
Fig. 12. (a) Fluorescence emission spectra showing the encapsulation
of Rhodamine B dye by the vesicles of peptide I in methanol-water and
(b) UV absorption spectra showing the encapsulation of Acridine Orange
dye by peptide I.
3. N. Nath and A. Chilkoti, Adv. Mater. 14, 1243 (2002).
4. B. Ozbas, J. Kretsinger, K. Rajagopal, J. P. Schneider, and D. J.
Pochan, Macromolecules 37, 7331 (2004).
5. C. J. Bosques and B. Imperiali, J. Am. Chem. Soc. 125, 7530 (2003).
J. Nanosci. Nanotechnol. 11, 6747–6756, 2011
6755

Salts Responsive Nanovesicles Through ꢀ-Stacking Induced Self-Assembly of Backbone Modified Tripeptides
Koley et al.
6. S. Murdan, J. Controlled Release 92, 1 (2003).
7. J. H. Collier, B.-H. Hu, J. W. Ruberti, J. Zhang, P. Shum, D. H.
Thompson, and P. B. Messersmith, J. Am. Chem. Soc. 123, 9463
(2001).
8. A. F. Dexter, A. S. Malcolm, and A. P. J. Middelberg, Nat. Mater.
5, 502 (2006).
34. S. Ghosh, S. K. Singh, and S. Verma, Chem. Commun. 22, 2296
(2007).
35. S. Ghosh, P. Singh, and S. Verma, Tetrahedron 64, 1250 (2008).
36. K. B. Joshi and S. Verma, Angew. Chem. Int. Ed. 47, 2860 (2008).
37. S. Aravinda, N. Shamala, R. Rajkishore, H. N. Gopi, and P. Balaram,
Angew. Chem. Int. Ed. 41, 3863 (2002).
9. J. R. Lu, S. Perumal, I. Hopkinson, J. R. P. Webster, J. Penfold,
W. Hwang, and S. Zhang, J. Am. Chem. Soc. 126, 8940 (2004).
10. Y. Tachibana, M. Kurisawa, H. Uyama, and S. Kobayashi, Biomacro-
molecules 4, 1132 (2003).
38. C. A. Hunter, K. R. Lawson, J. Perkins, and C. J. Urch, J. Chem.
Soc., Perkin Trans. 2, 651 (2001).
39. J. P. Gallivan and D. A. Dougherty, Proc. Natl. Acad. Sci. USA
96, 9459 (1999).
11. W. Frey, D. E. Meyer, and A. Chilkoti, Adv. Mater. 15, 248 (2003).
12. F. Checot, S. Lecommandoux, Y. Gnanou, and H.-A. Klok, Angew.
Chem. Int. Ed. 41, 1339 (2002).
13. A. K. Barman and S. Verma, Chem. Commun. 46, 6992 (2010).
14. E. G. Bellomo, M. D. Wyrsta, L. Pakstis, D. J. Pochan, and T. J.
Deming, Nat. Mater. 3, 244 (2004).
15. R. Fairman and K. S. Aakerfeldt, Curr. Opin. Struct. Biol. 15, 453
(2005).
16. X. Zhao and S. Zhang, Trends Biotechnol. 22, 470 (2004).
17. K. Rajagopal and J. P. Schneider, Curr. Opin. Struct. Biol. 14, 480
(2004).
40. M. Antonietti and S. Förster, Adv. Mater. 15, 1323 (2003).
41. S. Foster and M. Schmidt, Adv. Polym. Sci. 120, 51 (1995).
42. S. Forster, V. Abetz, and H. E. Muller, Advance in Polymer Science,
Springer, Berlin (2004), p. 173.
43. S. A. Jenekhe and X. L. Chen, Science 283, 372 (1999).
44. S. A. Jenekhe and X. L. Chen, Science 279, 1903 (1998).
45. S. J. Holder, R. C. Hiorns, N. Sommerdijk, S. J. Willims,
R. G. Jones, and R. J. M. Nolte, Chem. Commun. 14, 1445
(1998).
46. M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis,
Springer: New York (1984), p. 1.
18. A. Mishra, J. J. Panda, A. Basu, and V. S. Chauhan, Langmuir
24, 4571 (2008).
47. A. K. Das, A. Banerjee, M. G. B. Drew, S. Ray, D. Halder, and
A. Banerjee, Tetrahedron 61, 5027 (2005).
19. J. Naskar and A. Banerjee, Chem. Asian J. 4, 1817 (2009).
20. M. Reches and E. Gazit, Nano Lett. 4, 581 (2004).
21. P. P. Bose, A. K. Das, R. P. Hegde, N. Shamala, and A. Banerjee,
Chem. Mater. 19, 6150 (2007).
22. S. Santoso, W. Hwang, H. Hartman, and S. Zhang, Nano Lett. 2, 687
(2002).
48. CrysAlis, Oxford Diffraction Ltd., Abingdon, UK (2006).
49. G. M. Sheldrick, Shelxs97, and Shelxl97, Acta Cryst. A 64, 112
(2008).
50. P. Pandit, N. Chatterjee, S. Halder, S. K. Hota, A. Patra, and D. K.
Maiti, J. Org. Chem. 74, 2581 (2009).
51. W. Yin and M. Z. Yates, Langmuir 24, 701 (2008).
52. L. M. Dell’Acqua-Bellavitis and R. W. Seigel, Langmuir 24, 957
(2008).
23. R. Jayakumar, M. Murugesan, and M. R. Ahmed, Bioorg. Med.
Chem. Lett. 10, 1547 (2000).
24. S. Vauthey, S. Santoso, H. Gong, N. Watson, and S. Zhang, Proc.
53. S. Ghosh and S. Verma, Tetrahedron 64, 6202 (2008).
54. R. H. Schmidt and K. Haupt, Chem. Mater. 17, 1007 (2005).
55. R. H. Schimdt, K. Mosbach, and K. Haupt, Adv. Mater. 16, 719
(2004).
Delivered by Publishing Technology to: University of Waterloo
Natl. Acad. Sci. USA 99, 5355 (2002).
25. J. N. Israelachvili, D. J. Mitchell, and B. W. Ninham, Biochim Bio-
IP: 66.109.184.11 On: Fri, 30 Oct 2015 10:26:45
Copyright: American Scientific Publishers
phys Acta. 470, 185 (1977).
26. W. Cai, G.-T. Wang, Y.-X. Xu, X.-K. Jiang, and Z.-T. Li, J. Am.
Chem. Soc. 130, 6936 (2008).
56. I. Alfonso, M. Bru, M. I. Burguete, E. G. Verdugo, and S. V. Luis,
Chem. Eur. J. 16, 1246 (2010).
27. L.-Y. You, X.-K. Jiang, and Z.-T. Li, Tetrahedron 65, 9494 (2009).
28. C. Wang, S. Yin, S. Chen, H. Xu, Z. Wang, and X. Zhang, Angew.
Chem. Int. Ed. 47, 9049 (2008).
29. A. Ajayaghosh, P. Chithra, and R. Varghese, Angew. Chem. Int. Ed.
46, 230 (2007).
57. J. Han, G. Song, and R. Guo, Chem. Mater. 19, 973 (2007).
58. E. Gazit, FASEB J. 16, 77 (2002).
59. J. K. Lee and B. R. Lentz, Biochemistry 36, 6251 (1997).
60. R. J. Mart, R. D. Osborne, M. M. Stevens, and R. V. Ulijn, Soft
Matter. 2, 822 (2006).
30. X. Yan, Y. Cui, Q. He, K. Wang, J. Li, W. Mu, B. Wang, and Z.-C.
Ou-Yang, Chem. Eur. J. 14, 5974 (2008).
61. R. A. Curtis, J. M. Prausnitz, and H. W. Blanch, Biotechnol Bioeng.
57, 11 (1998).
31. B.-S. Kim, D.-J. Hong, J. Bae, and M. Lee, J. Am. Chem. Soc.
127, 16333 (2005).
62. S. Ghosh, M. Reches, E. Gazit, and S. Verma, Angew. Chem. Int.
Ed. 46, 2002 (2007).
32. S. Mukhopadhyay, U. Maitra, I. G. Krishnamoorthy, J. Schmidt, and
Y. Talmon, J. Am. Chem. Soc. 126, 15905 (2004).
33. A. Ajayaghosh, S. J. George, and V. K. Parveen, Angew. Chem. Int.
Ed. 42, 332 (2003).
63. P. Mukherjee, M. G. B. Drew, and A. Ghosh, Inorg. Chem. 48, 2364
(2009).
64. V. A. Klyachko and B. Jackson, Nature 418, 89 (2002).
65. W. A. Braell, Proc. Natl. Acad. Sci. USA 84, 1137 (1987).
Received: 2 December 2010. Accepted: 11 January 2011.
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