Fabrication of nanostructures through molecular self-assembly of small amphiphilic glyco-dehydropeptides

Article abstract of DOI:10.1039/c2mb25023c

Self-assembled peptide-based nanostructures have been the focus of research in the past decade because of their potential applications in various biological systems. Normally, small self-assembled peptide nanostructures contain hydrophobic moieties, there

Full text of DOI:10.1039/c2mb25023c

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Molecular
BioSystems
Cite this: Mol. BioSyst., 2012, 8, 1742–1749
www.rsc.org/molecularbiosystems
PAPER
Fabrication of nanostructures through molecular self-assembly of small
amphiphilic glyco-dehydropeptides
Manohar Mahato, Varun Arora, Rajiv Pathak, Hemant K. Gautam and
Ashwani Kumar Sharma*
Received 20th January 2012, Accepted 13th March 2012
DOI: 10.1039/c2mb25023c
Self-assembled peptide-based nanostructures have been the focus of research in the past decade
because of their potential applications in various biological systems. Normally, small self-assembled
peptide nanostructures contain hydrophobic moieties, therefore, their solubility in aqueous
systems poses the important challenge in the field of molecular self-assembly in order to make
effective use of these in a wide variety of applications. To improve their aqueous solubility, the
self-assembled amphiphilic a,b-dehydrophenylalanine containing small glyco-dehydropeptides,
Boc-Phe-DPhe-eAhx-GA (I) and H-Phe-DPhe-eAhx-GA (II) with glucosamine (GA) attached at
the C-terminal through a 6-aminocaproic acid linker, were synthesized, demonstrating the
formation of nanostructures in aqueous media, which were characterized by DLS, AFM and
TEM. Further, nanostructure II reduced auric chloride to gold nanoparticles and formed a
peptide–gold conjugate (VII). The feasibility of using the nanostructures I and II as nanovectors
for drug delivery was demonstrated by loading hydrophobic molecules, eosin and N-fluoresceinyl-
2-aminoethanol (FAE) dyes. Besides, these peptides displayed antimicrobial activity against
Micrococcus flavus, Bacillus subtilis and Pseudomonas aeruginosa. All these results advocate the
potential of these nanostructures as efficient vectors for drug delivery applications.
biomimetic confined nucleation of inorganic phases, in fabrica-
Introduction
tion of contact lenses, dye adsorbing agents, as well as adsorbents
for carcinogens.7 Hydrogels are generally composed of high
Recently, self-assembly has become a powerful approach
for fabricating novel nanomaterials through non-covalent
molecular weight natural polymers such as polysaccharides and
polypeptides, or synthetic polymers.4 Large polymer-based
interactions. Molecular self-assembly provides a route for
‘‘bottom-up’’ fabrication of remarkable functional nanoscale
hydrogels have been found to be useful, however, lack certain
merits possessed by the low molecular weight hydrogels.5 Cost
structures from simple molecular building blocks. The formation
of well-ordered structures by self-assembly of nucleic acids,
effectiveness, ease of synthesis, rapid response to external stimuli,
phospholipids, and polypeptides has attracted considerable
thermoreversibility, and low bioaccumulation are some of the
interest in the recent years.1–4 Besides the formation of interesting
important features of such hydrogels.
structures at the nanoscale, the phenomenon of self-assembly
In the last few years, we have seen tremendous development
has also been used for the generation of nanostructured
in the area of peptide-based nanostructures that has opened
the newer avenues for numerous applications.3,8,9 As peptides
materials that extend over macroscopic length scales as in
supramolecular gels. Though the gelation of polymers in
and peptide-derived conjugates offer novel scaffolds for con-
aqueous and organic media is well-documented, the design
venient design of various biomimetic materials, these have also
been shown to assemble into hydrogels.5 Several researchers
of stable hydrogelators has also been under considerable
focus.5,6 Hydrogels are three-dimensional networks capable
have reported the ability of dipeptides and tripeptides to self-
of imbibing large amount of water. These have been extensively
assemble and highlighted the usefulness of self-organized
structures.10–13 However, it is required to assess the stability
used as encapsulation matrices for controlled drug release,
scaffolds for tissue growth, repair and wound healing, micro-
of peptide-based structures under different physical, chemical,
fluidic devices as sensitive channel dams, as active sites for
and biochemical conditions to exploit them as useful bio-
molecular entities. In this context, recently, self-assemblies of
small peptides containing a,b-dehydrophenylalanine have
been shown to be stable at a broader range of pH conditions
CSIR-Institute of Genomics and Integrative Biology,
Delhi University Campus, Mall Road, Delhi 110 007, India.
E-mail: ashwani@igib.res.in; Fax: +91 11 27667471;
Tel: +91 11 27662491
as well as against proteases.14–16 Further, these assemblies
have displayed entrapment and release of bioactive molecules
c
1742 Mol. BioSyst., 2012, 8, 1742–1749
This journal is The Royal Society of Chemistry 2012

View Article Online
in a controlled manner. However, precise morphological control
and lack of solubility in aqueous systems have posed challenges
in the field of molecular self-assembly in order to use them
effectively in a wide variety of applications. The factors affecting
the shape, size and properties still require better insight in the
relationship between morphology, molecular organizations and
aspects of the self-assembly mechanisms.
peptides and their analogues in aqueous media has hampered
several potential applications and studies aiming at under-
standing the mechanism of the self-assembly at the molecular
level. In the present investigation, we have tried to address this
concern by synthesizing the amphiphilic glycolated dehydro-
peptides, I and II, following the standard solution phase
peptide synthesis method.14 Boc-Phe-DPhe-azlactone was
prepared from Boc-Phe-OH and DL-b-Pheser-ONa as shown
in Scheme 1. Glucosamine was conjugated via a 6-aminocaproic
acid (eAhx) linker to the C-terminus of the peptide. The GA
conjugated peptide was purified by RP-HPLC and characterized
by UV, 1H-NMR and mass spectroscopy. Incorporation of the
a,b-dehydrophenylalanine residue (DPhe) in these peptides
was confirmed by the presence of an intense absorption band
at 275 nm, a characteristic band due to the DPhe residue
(cinnamoyl moiety, C6H5–CQC–CQO) involving charge-
transfer electronic transition from the electron donating styryl
group to the electron accepting carbonyl group.16
Here, we report a preparation of an a,b-dehydrophenylalanine
residue (DPhe) containing amphiphilic glycol-dehydropeptides,
Boc-Phe-DPhe-eAhx-GA (I) and H-Phe-DPhe-eAhx-GA (II),
which self-assembled into stable nanostructures. The introduction
of DPhe, an unsaturated analog with a double bond between a
and b carbon atoms of the naturally occurring Phe,16 induced
not only conformational constraints in the peptide backbone
but also imparted proteolytic stability to the peptide. Incorporation
of glucosamine (GA) at C-termini via 6-aminocaproic acid
(eAhx), as a linker, provided the hydrophilicity to the designed
peptides, which helped in improving their solubility in aqueous
systems. Size and morphology of the synthesized peptides were
studied by DLS, AFM and TEM. Besides, these nanostructures
showed antimicrobial activity and were used to load hydrophobic
molecules. In addition, peptide II was used to reduce auric
chloride into gold nanoparticles, where it provided stabilization
to the particles so formed, as examined by TEM and AFM.
Self-assembly of the amphiphilic peptides
Self-assembly of the peptides was initiated by dispersing
peptide I or II (B1 mg) in a small volume of methanol (100 ml).
Subsequent addition of water (900 ml) with vigorous stirring
resulted in the formation of nanostructures, which was confirmed
by DLS measurements (Table 1). The size of peptide nano-
structures I and II was found to be B197 nm and B235 nm,
respectively. The solution containing higher concentration of
protected peptide I (10–20 mg mlÀ1) showed gel formation after
2–3 days, while the solution containing 1 mg mlÀ1 of peptide did
not form gel even after several weeks. Size and morphology of
these gels were further measured by AFM (Fig. 1). Self-
assembled peptides I and II displayed spherical particles with
size in the range of B55 nm and B175 nm, respectively. The
smaller size of these nanostructures in AFM compared to DLS
Results and discussion
In contrast to the complexity of natural supramolecules, the
self-assembled small molecules prepared by synthetic methods
have attracted much attention in recent years. Small and
modified peptides have been reported to self-assemble through
various interactions into shape-specific nanostructures, such as
micelles, vesicles, nanospheres, nanotubes, and many other
morphologies.17 The low solubility of the small self-assembled
Scheme 1 Schematic representation of synthesis of peptides I and II. Reaction conditions: (a) NMM/IBCF, ice–salt bath; (b) DL-b-Pheser-ONa;
(c) acidified to pH 2.0; (d) anhydrous CH3COONa/(CH3CO)2O; (e) HClÁeAhx-OMe/TEA; (f) 1 M NaOH/MeOH followed by acidification to
pH 2.0; (g) NMM/IBCF/NHS, ice–salt bath; (h) GAÁHCl/TEA; (i)TFA/DCM.
c
This journal is The Royal Society of Chemistry 2012
Mol. BioSyst., 2012, 8, 1742–1749 1743

View Article Online
Table 1 DLS measurements of self-assembled nanostructures
how the individual molecules assemble to form these nano-
structures, is yet to be understood. Since the aggregation process
in aqueous media is predominantly driven by the hydrophobic
effect, it would be interesting to monitor the assembly process
using loading of hydrophobic molecules such as eosin and FAE.
Eosin loaded nanostructures were prepared by dissolving the
peptides, I (5 mg), and eosin (1 mg) in 100 ml of MeOH and then
900 ml of water was added with continuous vortexing for 1 min.
The solutions were left overnight for stabilization of self-
assembled nano-structures and the solvent was removed in a
speed vac. Interestingly, the solubility of the peptide, III, improved
significantly after loading of the hydrophobic molecule, eosin,
which indicated that the hydrophobic molecule interacted with
the hydrophobic portion of self-assembled nanostructures and
helped in arrangement of nanostructures so that the hydrophilic
portion pointed outwards, while the hydrophobic portion got
buried inside of nanostructures to form nanovesicle-like nano-
structures, as shown in confocal microscopy images (Fig. 2a).
Likewise, FAE was loaded in peptides, I and II, to give FAE
loaded peptides, V and VI, respectively, and their confocal micro-
scopy images were taken, which showed a similar type of nano-
structures (Fig. 2b and c). Formation of nanovesicles was
supported by the TEM image of FAE loaded peptide, V
(Fig. 3b). From TEM images of unloaded peptide, I (Fig. 3a),
and FAE loaded peptide, V (Fig. 3b), it was observed that the
patterns of formation of nanostructures were different.
S. No.
Sample (1 mg mlÀ1
)
Size (nm) Æ SD
PDIa Æ SD
1.
2.
3.
4.
5.
6.
7.
I
II
197.13 Æ 3.5
235.2 Æ 4.2
172.73 Æ 2.4
135.46 Æ 1.4
360.15 Æ 15
340 Æ 20
0.195 Æ 0.013
0.351 Æ 0.032
0.183 Æ 0.012
0.384 Æ 0.028
0.45 Æ 0.025
0.57 Æ 0.031
0.45 Æ 0.038
III (I + eosin)
IV (II + eosin)
V (I + FAE)
VI (II + FAE)
VII (II–gold)
430 Æ 25
a
PDI: polydispersity index is a measure of the width of the particle
size distribution.
studies might be due to measurement of hydrodynamic diameter
in DLS, which measured the size of swollen nanostructures. The
average sizes of the loaded nanostructures upon addition of a
hydrophobic molecule, eosin, are shown in Table 1. Decrease in
the average size of loaded nanostructures was observed in
comparison to the unloaded nanostructures. This is apparently
due to greater propensity of the hydrophobic moieties to form
aggregates with compact hydrophobic pockets inside the self-
assembled network, which resulted in the hydrophobic–
hydrophobic interactions between eosin and hydrophobic moieties
inside the nanostructures.18,19 The electron micrograph showed
that the nanoparticles were composed of a highly dense network
of fibers. Nanoparticles were found to be spherical in shape. These
studies suggest that introduction of conformation constraint with
combination of hydrophilic moieties can be used as a strategy for
design of novel mesoscopic architectures.
Loading of dye molecules was also observed by eosin-
fluorescence quenching study. For this purpose, a solution of
peptide, I, in methanol was mixed with an aqueous solution of
eosin (10 mM in water) and the emission of eosin (lex, 515 nm;
Loading studies
lem, 540 nm) was monitored at different intervals of time
(Fig. 4). It was observed that with increasing time intervals,
there was a gradual drop in the fluorescence counts at 540 nm.
These results thus indicated that loading of eosin occurred via
hydrophobic interactions between dye molecules and the
hydrophobic moiety in peptide nanostructures.18,19
Loading of molecules in nanostructures is of primary importance
for their development as delivery vehicles. Different types of
weak interactions such as electrostatic, hydrophobic and
H-bonding have been found to be responsible for the formation
of self-assembled nanostructures but the actual mechanism,
Fig. 1 AFM images of (a) I, prepared in methanol : water (1 mg mlÀ1; 1 : 9, v/v); (b) II, prepared in water (1 mg mlÀ1); (c) VII, prepared in water
(1 mg mlÀ1). TEM images (without staining) of (d) II, prepared in water (1 mg mlÀ1); (e) VII, prepared in water (1 mg mlÀ1). The arrows represent
the gold particles in peptide-stabilized gold nanoparticles. (f) I, gel formation in methanol : water (5 mg in 0.4 ml; 1 : 1, v/v).
c
1744 Mol. BioSyst., 2012, 8, 1742–1749
This journal is The Royal Society of Chemistry 2012

View Article Online
Fig. 4 Fluorescence spectra showing quenching of eosin with peptide I.
Peptide I in eosin solution (10 mM) was excited at 515 nm and emission
fluorescence spectra were monitored in the range of 520–560 nm at
different time intervals.
Fig. 2 Confocal images of loaded peptides. (a) Eosin loaded peptide I
(III); (b) FAE loaded peptide I (V); (c) FAE loaded peptide II (VI).
Images were captured using rhodamine (for eosin), fluorescein (for FAE)
filters, DIC and overlaid.
structure with nanometre size particles/architecture. Addition
of eosin and FAE, used as models for hydrophobic cargo
molecules, to the nanostructure I in 10% MeOH followed by
lyophilisation rendered this peptide water soluble along with
eosin and FAE. CD spectra of these complexes, III (eosin
loaded) and V (FAE loaded) in water, were recorded, which
showed a strong change in 280 nm CD band characteristics.
These results revealed interactions of these molecules with
peptide I, which might help in loading in nanostructure I via
hydrophobic interactions. Interaction of eosin with peptide, I,
was also monitored by following the emission fluorescence
quenching (Fig. 4). It was found that eosin fluorescence at
B540 nm got quenched in a time dependent manner when
measured in 10% MeOH. Interaction of eosin with peptide II
was also observed with similar results in CD spectra. Peptide
II does not have a BOC group and is less hydrophobic as
compared to peptide I, therefore, became water soluble. It was
interesting to see that peptide nanostructure II adopted a more
ordered structure in water as observed in the CD spectrum
(Fig. 5b) compared to nanostructure I. As a negative CD band
at B280 nm indicated ordered state of DPhe containing
peptides, it became more pronounced when peptide II was
analysed in 100% MeOH suggesting highly ordered confor-
mation. Similarly, addition of FAE displayed similar effects
resulting in decreased ellipticity, B280 nm. These CD results
unequivocally demonstrated the interaction of eosin/FAE
with nanostructures I and II, which need to be further
investigated to see the possible use of nanostructures I and
II as delivery systems.
Circular dichroism (CD) spectroscopy
Loading of a cargo molecule, here, eosin and FAE, in a
nanostructure can be achieved if it interacts with the consti-
tuent molecules in the nanostructure. Presently, nano-
structures I and II have the DPhe amino acid residue, which
is known to give a characteristic CD band in the near UV
region of CD spectrum that strongly depends on the peptide
conformation.14,15 Therefore, the CD band of DPhe can serve
as a sensitive conformational probe to follow the interaction of
cargo molecules with nanostructures having DPhe residues.
We recorded the CD spectra of nanostructures I and II in the
UV range of 240–320 nm (Fig. 5a and b), however, inter-
actions could be detected from the near UV CD spectrum, as
in the far UV spectrum, band assignments become difficult due
to overlapping contribution of peptide and aromatic chromo-
phores. Fig. 5a depicts the CD spectra of nanostructure I in
100% MeOH, nanostructure I in 10% MeOH in water,
nanostructure III in water and nanostructure V in water.
In 100% MeOH, the CD spectrum of I consisted of a well-
defined negative CD trough at around 280 nm, which got
pronounced further when we analysed I in 10% MeOH, a
condition in which peptide I formed nanostructures as shown
in DLS and AFM. Increase in negative ellipticity of the CD
band, which was due to the DPhe side chain, demonstrated a
more ordered structure of peptide I, which could lead to
organisation of this dipeptide moiety into a supramolecular
Fig. 3 TEM images (negative staining with 1% uranyl acetate) of (a) peptide I and (b) I + FAE, V.
c
This journal is The Royal Society of Chemistry 2012
Mol. BioSyst., 2012, 8, 1742–1749 1745

View Article Online
Table 2 Zone of inhibition (mm) against several pathogenic microbial
test strains caused by peptides I and II, exhibiting their antibacterial
activity
Diameter of zone of inhibitiona/mm
I
II
10% methanolb
Microbial test strains A B C A B C in water
Gentamicinc
18
Micrococcus flavus
(NCIM 2378)
Staphylococcus aureus 7
7
7
7
9 7 10 10
8 7
—
6
7
—
—
15
19
Bacillus subtilis
(NCIM-2063)
7 8 10 7 9 10
Pseudomonas aeruginosa 7
(NCIM 2053)
7
8
9 7
8 7
9
7
9
7
—
—
17
16
Escherichia coli
(NCIM 2739)
8
a
Diameter of zone of inhibition (mm) including disk diameter of
6 mm; A, B and C: concentration of compounds in methanol coated
on each disk (100 mg per disk, 500 mg per disk, 1000 mg per disk,
a negative control.
b
respectively). 10% methanol in water as
c
Gentamicin (10 mg per disk) was used as positive reference standard
antibiotic disk; (—): no activity detected.
and membrane disruption is understandable based on the
structure of the antibacterial peptides. These peptides comprise
hydrophilic and strongly positively charged domains, which
are proposed to initiate peptide interaction with the negatively
charged bacterial surface and the negatively charged head
groups of bilayer phospholipids.24,25 Recently, a number of
cationic and amphipathic dehydrophenylalanine containing
peptides and glycopeptide derivatives have been shown to
possess antibacterial activity.26,27 Here antimicrobial activity
tests with peptides, I and II, were performed by using disk
diffusion assay.28 Three test strains of Gram-positive bacteria,
Micrococcus flavus, Bacillus subtilis and Staphylococcus
aureus, and two test strains of Gram-negative bacteria,
Escherichia coli and Pseudomonas aeruginosa, were used to
investigate antibacterial activity of the peptides. The anti-
bacterial activity of both peptides against different test organisms
is shown in Table 2. Both peptides showed good antibacterial
activity against Micrococcus flavus, Bacillus subtilis and
Pseudomonas aeruginosa. These findings may serve as a
footstep regarding the biological and pharmacological activities
of these peptides.
Fig. 5 Circular dichroism (CD) spectroscopy of peptides.
Preparation of peptide-stabilized gold nanoparticles
Peptide nanostructures offer surfaces that can be chemically
functionalized for building biosensors, and/or can serve as
organic templates for the growth of ordered inorganic phases,
leading to applications in nanotechnology.3,20,21 Peptide II
was used for the preparation of peptide-stabilized gold nano-
particles. These nanoparticles were prepared by refluxing a
solution of peptide II and HAuCl4 (0.01%) for 3 h, which led
to the formation of a dark, violet-coloured solution indicating
the reduction of HAuCl4 to gold nanoparticles. Appearance of
an absorption band at 555 nm due to surface plasmon
resonance (SPR) confirmed the preparation of gold nano-
particles. DLS study of these stabilized gold nanoparticles
VII showed an average particle size of 430 nm (Table 1). While
the morphology of these nanostructures was studied by AFM
(Fig. 1c), which displayed these particles as spherical in shape.
Ultrastructure of a peptide–gold nanoparticle was character-
ized by TEM (Fig. 1e), which confirmed the spherical shape
of the stabilized gold nanoparticle. Similar structures have
also been observed with other peptide-based systems, as
reported in the literature.22,23 Thus, unlike larger peptides,
self-assembly in small peptides appears not to be governed
by the molecular structure of the peptide alone, but also
depends on the conditions employed during the assembly.
Further conjugation of these peptide–gold nanoparticles with
thiolated ligands would be quite useful for various biological
applications.
Antimicrobial activity
Fig. 6 Cell viability of peptides in HeLa cells. For solvent controls, cells
were treated with DMEM media containing 10% FBS (M), cells were
treated with water for peptide II and with 1% DMSO for peptide I.
Generally, antimicrobial peptides interact with the bacterial
cell membrane. A direct correlation between antibiotic effect
c
1746 Mol. BioSyst., 2012, 8, 1742–1749
This journal is The Royal Society of Chemistry 2012

View Article Online
Cytotoxicity assay
Boc-Phe-DPhe-azlactone
MTT assay involves the reduction of yellow MTT by mito-
chondrial succinate dehydrogenase in live cells into a colored
(dark purple) formazan product. The formazan crystals so
formed are solubilized in organic solvents, and intensity is
measured spectrophotometrically at 540 nm. The results
indicated that HeLa cells showed more than 95% viability
up to 25 mM concentration of peptides I and II (Fig. 6).
To a pre-cooled solution of Boc-Phe-OH (4.0 g, 15 mmol)
containing NMM (2.2 ml, 20 mmol) in dry tetrahydrofuran
(THF) (20 ml), in an ice–salt bath, was added isobutyl
chloroformate (IBCF) (2.6 ml, 20 mmol) dropwise. After 5 min,
a pre-cooled aqueous solution of DL-b-Pheser (3.6 g, 20 mmol)
containing sodium carbonate (1.1 g, 10 mmol) was added to
the above reaction mixture and the reaction was stirred at the
same temperature for 2 h. After completion of reaction
(as monitored on tlc), the reaction mixture was concentrated
on a rotavapor to remove the organic solvent. The remaining
aqueous phase was washed with ethyl acetate (3 Â 20 ml). The
aqueous phase was collected and acidified with a 10% aqueous
solution of citric acid to pH 2.0. The product was extracted
with ethyl acetate (3 Â 20 ml) and the organic phase was finally
washed twice with water (2 Â 15 ml) and dried over anhydrous
sodium sulfate. After filtration, the solvent was removed
in vacuo to give an oily compound, Boc-Phe-DL-b-Pheser,
which was treated with anhydrous sodium acetate (1.0 g) in
acetic anhydride (10 ml) and the resulting reaction mixture was
left overnight at room temperature. Crushed ice was then added
to the slurry and the compound was kept at 4 1C for 24 h. Water
was decanted off and the oily compound was redissolved in
ethyl acetate and washed successively with a 10% aqueous
solution of NaHCO3 (3 Â 15 ml) and cold water (3 Â 15 ml),
and finally dried over anhydrous sodium sulfate. After filtration,
the solvent was removed in vacuo to give an oily compound,
Boc-Phe-DPhe-azlactone, 2.31 g (B34% yield).
Conclusions
An easy and cost-effective synthesis of self-assembled amphi-
philic glyco-dehydropeptides with improved solubility in
aqueous medium has been demonstrated. We have shown the
ability of these amphiphilic peptides to form hydrogels at higher
concentration and nanostructures at lower concentration.
Amphiphilic peptide II with a free amino group efficiently
reduced auric chloride to gold nanoparticles as well as provided
stability to these nanoconjugates. Interestingly, these self-
assembled peptides displayed antimicrobial activity against
Micrococcus flavus, Bacillus subtilis and Pseudomonas aeruginosa.
Moreover, these amphiphilic peptides have been shown to
possess capability of loading hydrophobic molecules. These
glycopeptide-based nanostructures can be applied as drug
delivery nanocarriers and hydrogels for sustained drug release
and tissue engineering, bearing antibacterial activity, and as
scaffolds to present multivalently complex carbohydrates.
Methods
Boc-Phe-DPhe-eAhx-OMe
Materials
To a solution of eAhx-OMeÁHCl (1.07 g, 5.9 mmol) and
triethylamine (TEA) (0.81 ml, 5.9 mmol) in dry THF, Boc-
Phe-DPhe-azlactone (2.31 g, 5.9 mmol) was added and the
reaction mixture was stirred overnight at room temperature.
After completion of reaction, the solvent was removed
in vacuo. The residue was dissolved in ethyl acetate (20 ml)
and washed sequentially with a solution of 10% citric acid
(3 Â 15 ml), water (2 Â 20 ml), 10% aqueous solution of
NaHCO3 (3 Â 20 ml) and water (2 Â 15 ml). The organic
phase was collected, dried over anhydrous sodium sulfate,
filtered and concentrated in vacuo to yield a crude product,
Boc-Phe-DPhe-eAhx-OMe, which was purified by column
chromatography using a gradient of methanol–dichloroethane.
The pure fractions containing the compound were pooled together
and concentrated in vacuo to obtain the final compound, Boc-Phe-
DPhe-eAhx-OMe, 2.2 g (B65% yield). UV, lmax: 274 nm (DPhe).
1H NMR (CDCl3, 400 MHz) d: 7.28–7.34 (10H, m, Ar–H),
3.65 (3H, s, –OCH3), 2.95 (2H, m, –N–CH2–), 2.32 (2H, t,
–COCH2–), 1.58 (4H, m, –CH2– Â2), 1.39 (9H, s, Boc), 1.24
(2H, m, –CH2–).
N-Methylmorpholine (NMM), fluorescein isothiocyanate (FITC),
isobutylchloroformate (IBCF), N-hydroxysuccinimide (NHS),
Boc-Phe-OH, DL-b-phenylserine (DL-b-Pheser), D(+)-glucosamine
(GA) HCl and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) were obtained from Sigma-
Aldrich Co., USA. 6-Aminocaproic acid (eAhx) was obtained
from Fluka AG, Switzerland. All other chemicals and solvents
used were of analytical grade and procured locally.
Peptide synthesis
Amphiphilic peptides were synthesized by the standard solution
phase method.14 For analysis purpose, the peptides were purified
on a reverse phase C8 column (Zorbax eclipse XDB-C8 4.6 Â
150 mm, 5 mm) using a gradient of acetonitrile–water (containing
0.01% TFA) on HPLC (Agilent 1100 series with a binary pump
system). Purified peptides were analysed by mass spectroscopy
(LCMS with triple quadrupole Quattro micro API, Waters Inc.
USA) and 1H-NMR (Bruker Spectrospin spectrometer, 400 MHz).
UV spectroscopy, circular dichroism (CD) and fluorescence
spectroscopy were carried out on a Lambda Bio 20 UV spectro-
photometer (Perkin Elmer, USA), a JASCO J-815 Spectro-
polarimeter (Jasco Corp., Tokyo, Japan) and a Spectrofluorometer
(Edinburgh Instruments, UK), respectively. For drug loading
study, N-fluoresceinyl-2-aminoethanol (FAE) was prepared by
reaction of fluorescein isothiocyanate (FITC) with ethanol-
amine. Confocal images were taken on a Zeiss LSM 510
inverted laser-scanning confocal microscope (UK).
Boc-Phe-DPhe-eAhx-OH
Boc-Phe-DPhe-eAhx-OMe (2.16 g, 4 mmol) was dissolved in
methanol (20 ml) and an aqueous solution of sodium hydroxide
(7.0 ml, 1 M) was added to hydrolyze the methyl ester. The
resulting mixture was stirred overnight at room temperature
and then it was partially concentrated to remove methanol.
The reaction mixture was diluted with water (20 ml) and washed
c
This journal is The Royal Society of Chemistry 2012
Mol. BioSyst., 2012, 8, 1742–1749 1747

View Article Online
with ethyl acetate (3 Â 30 ml). The aqueous phase was
collected and acidified with a 10% aqueous solution of citric
acid to pH 2.0 and the compound was extracted with ethyl
acetate (3 Â 20 ml). The combined organic phase was washed
twice with water (2 Â 20 ml), dried over anhydrous sodium
sulfate, filtered and solvent evaporated in vacuo to yield
Characterization of nanostructures
Self-assembled peptide nanostructures were characterized by
dynamic light scattering (DLS), atomic force microscopy
(AFM) and high resolution transmission electron microscopy
(HR-TEM). DLS studies were carried out on Zetasizer Nano-ZS
(Malvern Instruments, UK). The data analysis was performed in
automatic mode and data are presented as the average value of
20 runs. AFM studies were also carried out at 1 mg mlÀ1
concentration of peptides on a microscope (PicoSPM System,
Molecular Imaging, USA) in acoustic mode and images were
acquired using SPIP software. For TEM analyses, the samples
were prepared by depositing the solution of the nanostructures
(1 mg mlÀ1) on carbon-coated copper grids with and without
1% uranyl acetate negative staining and images were observed
at an accelerating voltage of 200 kV on HR-TEM (Technai G2
30U-twin, Technai 300 kV ultra twin microscope).
Boc-Phe-DPhe-eAhx-OH, 1.47 g (B70% yield). UV, lmax
273 nm (DPhe).
:
Boc-Phe-DPhe-eAhx-GA (I)
To a pre-cooled solution of Boc-Phe-DPhe-eAhx-OH (1.47 g,
2.8 mmol) and NMM (0.3 ml, 2.8 mmol) in dry THF (20 ml),
in an ice–salt bath, was added IBCF (0.4 ml, 2.8 mmol)
dropwise. After 5 min, a pre-cooled solution of NHS (0.35 g,
3 mmol) in THF (5 ml) was added and the resulting mixture
was stirred at room temperature for 1 h. Then an aqueous
solution of glucosamine hydrochloride (0.64 g, 3 mmol) con-
taining triethylamine (TEA) (1 ml) was added to the above
reaction mixture. After completion of reaction (B12 h), the
solvent was concentrated in vacuo and the desired compound
was extracted with ethyl acetate (3 Â 20 ml). The combined
organic phase was sequentially washed with water (2 Â 15 ml)
and saturated sodium chloride (2 Â 20 ml), dried over
anhydrous sodium sulfate, filtered and concentrated to yield
Circular dichroism (CD) spectroscopy
CD spectra of self-assembled peptide solutions were recorded
in the far UV range (240–320 nm) using a rectangular quartz
cuvette of 0.1 cm path length and 1 nm bandwidth at 0.1 nm
resolution at 0.2 mg mlÀ1 concentration. Each spectrum was
recorded as an average of three repeated scans in a continuous
scanning mode with 100 nm sÀ1 scanning speed and response
time of 1 second. The contribution of buffer/solvent was
subtracted from each spectrum. CD spectra of peptides were
recorded in different solvents and their mixtures. Information
was drawn mainly on phenylalanine side chain CD contribution
in the near UV region of CD spectra.
Boc-Phe-DPhe-eAhx-GA, I, 1.24 g (B65% yield). UV, lmax
:
275 nm (DPhe); MS: m/z, 684.43 (calculated) and 685.10
(observed) (M + H+).
TFAÁH-Phe-DPhe-eAhx-GA (II)
Loading studies
Removal of the Boc protecting group from I was carried out
by treatment with dichloromethane (DCM) : trifluoroacetic
acid (TFA) (1 : 1, v/v, 5 ml) for 3 h at room temperature. The
solvent was removed and the residue was precipitated with
diethyl ether. The resulting precipitate was filtered, washed
several times with dry ether, and dried to obtain compound II
as a TFA salt in quantitative yield. UV, lmax: 274 nm (DPhe).
MS: m/z, 584.3 (calculated) and 585.10 (observed) (M + H+).
Eosin-loaded nanostructures were prepared by dissolving the
peptides I/II (5 mg) and eosin (1 mg) in 100 ml MeOH followed
by the addition of 900 ml of water with continuous vortexing
for 1 min. The solutions were left overnight for stabilization of
self-assembled nanostructures, their mean size was measured
by DLS and solvent removed in a speed vac. Likewise, FAE
loaded nanostructures were prepared. To study the loading of
eosin molecules in nanostructures I, eosin quenching assay was
performed. An aqueous solution of eosin (900 ml, 10 mM) was
added to a solution of I (1 mg in 100 ml MeOH). Emission
fluorescence spectra were recorded in the range of 520–620 nm
at different time intervals with excitation at 515 nm. For
confocal study of loaded nanostructures, slides were prepared
using air dried solution (1 ml) of 0.2 mg mlÀ1 concentration of
loaded peptides III, V and VI.
Preparation of nanostructures
Self-assembly of the peptides was initiated by dispersing B1–5 mg
of the peptide I/II in a small volume of methanol (0–200 ml).
Water was added to make up the final volume (1 ml) with
vigorous stirring on a vortex shaker.
Preparation of peptide II-stabilized gold nanoparticles (VII)
Antimicrobial activity of peptides
Peptide II stabilized gold nanoparticles were prepared by
refluxing the aqueous solution of 0.01% HAuCl4 (20 ml)
and peptide II (0.4 ml, 1 mg mlÀ1). After 3 h, the colour of
the solution changed from yellowish to purple indicating
formation of gold nanoparticles. The UV absorption spectrum
of the resulting solution showed a peak near 555 nm. The
mixture was centrifuged at 12 000 rpm (2 Â 1 h). The super-
natant was decanted off and the nanoparticles were washed
twice with water and finally resuspended in 1 ml of water for
further analyses. Size of these nanoparticles was measured on
DLS and morphological studies were carried out using TEM.
Antimicrobial activity of self-assembled peptides, I and II, was
determined by using the Kirby–Bauer single disk susceptibility
test.28 The sterile filter paper disks of 6 mm diameter were
loaded with two different peptides, I and II, prepared in three
different concentrations in 10% methanol in water on each
disk (100 mg per disk, 500 mg per disk and 1000 mg per disk,
respectively). Filter paper disks soaked with each peptide were
placed in the respective grid of each Mueller Hinton agar plates
seeded with target test organisms and then kept for incubation at
37 1C overnight. The results were recorded by measuring the
c
1748 Mol. BioSyst., 2012, 8, 1742–1749
This journal is The Royal Society of Chemistry 2012

View Article Online
zone of inhibition surrounding the disk. A gentamicin disk
(10 mg per disk) and 10% methanol in water were used as positive
and negative controls, respectively, for bacterial test strains.
9 E. Gazit, Self Assembly of Short Aromatic Peptides into Amyloid
Fibrils and Related Nanostructures, Prion, 2007, 1, 32–35.
10 A. Mahler, M. Reches, M. Rechter, S. Cohen and E. Gazit, Rigid,
Self-Assembled Hydrogel Composed of a Modified Aromatic
Dipeptide, Adv. Mater., 2006, 18, 1365–1370.
11 S. Toledano, R. J. Williams, V. Jayawarna and R. V. Ulijn,
Enzyme-Triggered Self-Assembly of Peptide Hydrogels via Reversed
Hydrolysis, J. Am. Chem. Soc., 2006, 128, 1070–1071.
12 A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins,
M. L. Turner, A. Saiani and R. V. Ulijn, Fmoc-Diphenylalanine
Self Assembles to a Hydrogel via a Novel Architecture Based on
p–p Interlocked b-Sheets, Adv. Mater., 2008, 20, 37–41.
13 A. Mishra and V. S. Chauhan, Probing the Role of Aromaticity in
the Design of Dipeptide Based Nanostructures, Nanoscale, 2011, 3,
945–949.
14 J. J. Panda, A. Mishra, A. Basu and V. S. Chauhan, Stimuli
Responsive Self-Assembled Hydrogel of a Low Molecular Weight
Free Dipeptide with Potential For tunable Drug Delivery, Bio-
macromolecules, 2008, 9, 2244–2250.
15 A. Mishra, J. J. Panda, A. Basu and V. S. Chauhan, Nanovesicles
Based on Self-Assembly of Conformationally Constrained
Aromatic Residue Containing Amphiphilic Dipeptides, Langmuir,
2008, 24, 4571–4576.
Cytotoxicity assay
Cytotoxicity of peptides (I and II) was evaluated on HeLa cells
by MTT colorimetric assay. Cells were grown at B8000 cells
per well in a 96-well plate (in DMEM medium with 5% CO2 at
37 1C for 18 h). Cells were treated with peptides I and II at
different concentrations (10, 25, 50 and 100 mM) in 1% DMSO
and water respectively. Cells treated with complete media, 1%
DMSO and water were taken as controls. After 36 h, MTT
(200 ml, 0.5 mg mlÀ1 in DMEM) was added to cells followed
by incubation for 1 h. Formazan crystals were then dissolved
in 100 ml isopropanol containing 0.06 M HCl and 0.5% SDS.
The intensity of colour was measured spectrophotometrically
on an ELISA plate reader (MRX, Dynatech Laboratories) at
540 nm. The relative cell viability of treated samples was
calculated (%) by [abs]sample/[abs]control  100 by taking their
respective controls.
16 M. Gupta and V. S. Chauhan, De Novo Design of a,b-Didehydro-
phenylalanine Containing Peptides: from Models to Applications,
Biopolymers, 2011, 95, 161–173.
17 M. Reches and E. Gazit, Designed Aromatic Homo-Dipeptides:
Formation of Ordered Nanostructures and Potential Nanotechno-
logical Applications, Phys. Biol., 2006, 3, S10–S19.
Acknowledgements
18 A. Archut, G. C. Azzellini, V. Balzani, L. D. Cola and F. Vogtle,
Toward Photoswitchable Dendritic Hosts. Interaction between
Azobenzene-Functionalized Dendrimers and Eosin, J. Am. Chem.
Soc., 1998, 120, 12187–12191.
19 S. Patnaik, A. K. Sharma, B. S. Garg, R. P. Gandhi and
K. C. Gupta, Photoregulation of Drug release in Azo-Dextran
Nanogels, Int. J. Pharm., 2007, 342, 184–193.
20 M. B. Dickerson, K. H. Sandhage and R. R. Naik, Protein- and
Peptide-directed Syntheses of Inorganic Materials, Chem. Rev.,
2008, 108, 4935–4978.
The authors gratefully acknowledge the financial support from
the project EXP002. MM thanks the University Grants
Commission, New Delhi, India, for the award of Junior
Research Fellowship to carry out the projected work. The
authors also thank Dr M. Ganguli (IGIB) for AFM,
Dr Tapan K. Jain (IPU) and Mr Rahul Bhardwaj (USIC, DU)
for their help in TEM imaging.
21 C. L. Chen and N. L. Rosi, Peptide-based Methods for the
Preparation of Nanostructured Inorganic Materials, Angew.
Chem., Int. Ed., 2010, 49, 1924–1942.
References
1 I. Brigger, C. Dubernet and P. Couvreur, Nanoparticles in Cancer
Therapy and Diagnosis, Adv. Drug Delivery Rev., 2002, 54, 631–651.
2 S. Zhang, Fabrication of Novel Biomaterials through Molecular
Self-Assembly, Nat. Biotechnol., 2003, 21, 1171–1178.
3 E. Gazit, Self-Assembled Peptide Nanostructures: the Design of
Molecular Building Blocks and their Technological Utilization,
Chem. Soc. Rev., 2007, 36, 1263–1269.
22 J. Kim, Y. Rheem, B. Yoo, Y. Chong, K. N. Bozhilov, D. Kim,
M. J. Sadowsky, H. G. Hur and N. V. Myung, Peptide-mediated
Shape- and Size-tunable Synthesis of Gold Nanostructures, Acta
Biomater., 2010, 6, 2681–2689.
23 J. Kim, J. M. Sadowsky and H. G. Hur, Simultaneous Synthesis of
Temperature-tunable Peptide and Gold Nanoparticle Hybrid
Spheres, Biomacromolecules, 2011, 12, 2518–2523.
4 C. Goncalves, P. Pereira and M. Gama, Self-Assembled Hydrogel
Nanoparticles for Drug Delivery Applications, Materials, 2010, 3,
1420–1460.
5 M. de Loos, B. L. Feringa and J. H. van Esch, Design and
Application of Self-Assembled Low Molecular Weight Hydrogels,
Eur. J. Org. Chem., 2005, 3615–3631.
6 M. McCullagh, T. Prytkova, S. Tonzani, N. D. Winter and
G. C. Schatz, Modeling Self-Assembly Processes Driven by
Nonbonded Interactions in Soft Materials, J. Phys. Chem. B,
2008, 112, 10388–10398.
7 X. Yan, P. Zhu and J. Li, Self-Assembly and Application of
Diphenylalanine-Based Nanostructures, Chem. Soc. Rev., 2010,
39, 1877–1890.
24 L. J. Otvos, The Short Proline-rich Antibacterial Peptide Family,
Cell. Mol. Life Sci., 2002, 59, 1138–1150.
25 J. B. McPhee and R. E. W. Hancock, Function and Therapeutic
Potential of Host Defence Peptides, J. Pept. Sci., 2005, 11,
677–687.
26 P. C. Dewan, A. Anantharaman, V. S. Chauhan and D. Sahal,
Antimicrobial Action of Prototypic Amphipathic Cationic
Decapeptides and their Branched Dimers, Biochemistry, 2009, 4,
5642–5657.
27 L. Motiei, S. Rahimipour, D. A. Thayer, C. H. Wong and
M. R. Ghadiri, Antibacterial Cyclic D,L-Alpha-Glycopeptides,
Chem. Commun., 2009, 3693–3695.
28 R. Pathak, R. Singh, A. Singh, H. Gautam, A. S. Dhaker,
R. Kumar, R. Arora and H. K. Gautam, Assessment of Anti-
Bacterial and Free Radical Scavenging Activity in Psychrophilic
Arthrobacter sp., Pharmacologyonline, 2011, 1, 344–355.
8 S. Gilead and E. Gazit, Self-Organization of Short Peptide
Fragments: from Amyloid Fibrils to Nanoscale Supramolecular
Assemblies, Supramol. Chem., 2005, 17, 87–92.
c
This journal is The Royal Society of Chemistry 2012
Mol. BioSyst., 2012, 8, 1742–1749 1749