Mesoporous vesicles from supramolecular helical peptide as drug carrier

Article abstract of DOI:10.1039/c1sm05958k

The self-assembly of a hydrophobic pentapeptide has been investigated in an attempt to develop a drug delivery vehicle. The peptide forms a supramolecular helical column through intra- and intermolecular hydrogen bonding interactions and an interdigitated helical bundle structure in the solid state. In methanol solution, the peptide forms mesoporous vesicles, where the diameters of the vesicles vary with the concentration in direct proportion. The most important property of these mesoporous vesicular structures is the encapsulation of a potent bacteriostatic antibiotic, sulfamethoxazole. Moreover, at pH 6.2, the drug loaded vesicles can effectively release the encapsulated drug slowly, which holds future promise for use of the microvesicles as drug cargo.

Full text of DOI:10.1039/c1sm05958k

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PAPER
Mesoporous vesicles from supramolecular helical peptide as drug carrier†
*
Sibaprasad Maity, Poulami Jana, Suman Kumar Maity and Debasish Haldar
Received 24th May 2011, Accepted 5th August 2011
DOI: 10.1039/c1sm05958k
The self-assembly of a hydrophobic pentapeptide has been investigated in an attempt to develop a drug
delivery vehicle. The peptide forms a supramolecular helical column through intra- and intermolecular
hydrogen bonding interactions and an interdigitated helical bundle structure in the solid state. In
methanol solution, the peptide forms mesoporous vesicles, where the diameters of the vesicles vary with
the concentration in direct proportion. The most important property of these mesoporous vesicular
structures is the encapsulation of a potent bacteriostatic antibiotic, sulfamethoxazole. Moreover, at pH
6.2, the drug loaded vesicles can effectively release the encapsulated drug slowly, which holds future
promise for use of the microvesicles as drug cargo.
self-assembly of a hydrophobic pentapeptide to the fabrication
Introduction
of mesoporous microvesicles. From X-ray crystallography the
peptide forms a supramolecular helical column through intra-
and intermolecular hydrogen bonding interactions and an
interdigitated helical bundle structure, which may further self-
assemble to form the vesicles. These microvesicles can encapsu-
late a potent bacteriostatic antibiotic sulfamethoxazole and
slowly release the encapsulated drug at pH 6.2.
Self-assembled porous micro/nanostructure materials have
attracted increasing interest in their potential applications as
storage, efficient catalysts and delivery vehicles.1 These materials
have different compositions such as organic capsules,2 polymers,3
lipids,4 amino acid derivatives,5 peptides,6 silica,7 carbon,8
metals, metal oxides,9 or metal organic frameworks,10 which can
be constructed by employing various methods, like emulsions,
spray-drying, hydrothermal reduction, templating and layer-by-
layer assembly.11 In recent years, great progress has been made in
fabricating drug carriers such as microbubbles,12 liposomes,13
and nanoparticles.14 However, particle based drug delivery
systems offer many advantages compared to liposomes and
micelles,15 including the capacity to incorporate both small and
large molecules, hydrophobic and hydrophilic drugs, enhanced
stability, and enhanced drug carrying capacity.16 Peptide based
vesicles are very interesting due to their well-defined structure,17
molecular recognition properties,18 biocompatibility19 and
biodegradibility.20
Results and discussion
The pentapeptide Boc–Leu(1)–Aib(2)–Phe(3)–Phe(4)–Aib(5)–
OMe, 1, consisting of hydrophobic amino acid residues, has been
synthesized by conventional solution phase methodology and
purified, characterized and studied. There are two conforma-
tionally constrained a-aminoisobutyric acid (Aib) residues to
increase the helical nature of the peptide back bone and to
enhance the crystallinity.25 To assess the formation of self-
assembled structures, dynamic light scattering (DLS) experi-
ments were performed. DLS is a rapid scanning method used to
identify nanostructures by size distribution. To determine the
size of the nanostructure formed by the self-assembly of peptide
1, DLS experiments were performed at different concentrations
in methanol. From the DLS study, at a low concentration (0.5
mg mLꢀ1 in freshly prepared methanol solution), peptide 1 self-
assembled into nanosized structures of 255–400 nm (Fig. 1a),
whereas at a moderate concentration (1 mg mLꢀ1) the particle
size increased to 530–825 nm in diameter (Fig. 1b). However, at
a higher concentration (2 mg mLꢀ1), the diameters of the
self-assembled nanostructured particles increased to about 530
nm–1.4 mm (Fig. 1c). Hence the DLS experiments show that the
self-assembled particle size of peptide 1 gradually increases with
concentration in methanol.
We are involved in studying the self-assembly of short
synthetic peptides.21 Recently, we have reported the fabrication
of self-assembled peptide based nanoporous materials that can
be used as in situ reducing agents22 or that can remove organic
dyes from wastewater.23 Also we have described the formation of
microspheres by self-assembly of short peptides and the transi-
tion from a sphere to a rod structure.24 Herein, we present the
Department of Chemical Sciences, Indian Institute of Science Education
and Research, Kolkata, Mohanpur, West Bengal, 741252, India. E-mail:
deba_h76@yahoo.com; deba_h76@iiserkol.ac.in; Fax: +91-34-73
279131; Tel: +91-34-73 279130
† Electronic supplementary information (ESI) available: Synthesis and
characterization of peptides, 1H NMR, 13C NMR, Figures ESI S1–8,
Figures S1–S19. CCDC reference number 827205. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c1sm05958k
The morphologies of these self-assembled peptide nano-
structures were studied by field emission scanning electron
microscopy (FE-SEM) at different peptide 1 concentrations in
10174 | Soft Matter, 2011, 7, 10174–10181
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Fig. 3 (a) AFM images of microvesicles of peptide 1 from methanol at
0.5 mg mLꢀ1, (b) 3D image of peptide 1 vesicles and (c) height profile plot
of peptide 1 vesicle (line marked in a).
Fig. 1 DLS studies of peptide 1 self-assembly in methanol at concen-
trations: (a) 0.5 mg mLꢀ1, (b) 1 mg mLꢀ1 and (c) 2 mg mLꢀ1
.
the 3D images of peptide vesicles. From AFM, the calculated
height of the vesicles of peptide 1 was about 400 nm (Fig. 3c).
The stability of the microvesicles was further studied by TGA–
DSC experiments. Both the TGA curve and the DSC curve show
that no decomposition, endotherms or exotherms are obtained
befoꢁre 220 ꢁC and hence these vesicles are thermally stable up to
220 C (ESI Fig. 1 and 2†).
methanol. The FE-SEM images elucidated the formation of
microvesicles of various sizes depending on the concentration.
Fig. 2a shows that peptide molecules self-assembled to form
polydispersed spherical vesicles with a diameter range from 200–
500 nm at 0.5 mg mLꢀ1 in methanol. At a higher concentration of
1 mg mLꢀ1 the particle size increases to 400 nm–1.2 mm in
diameter (Fig. 2b). Fig. 2c exhibits the very large vesicles with
diameters of about 3.5 mm at a concentration of 5 mg mLꢀ1 in
methanol. The FE-SEM images show that the vesicles are mes-
oporous in nature (Fig. 2c, d).
To investigate the secondary structure of peptide 1 in vesicles,
solid state FT-IR spectroscopy was performed. The region 1800–
1500 cmꢀ1 is important for the stretching band of amide I and
bending peak of amide II.26 Another informative frequency range
is 3500–3200 cmꢀ1, corresponding to the N–H stretching vibra-
tions.27 In comparison with the as synthesized compound, in
vesicles the FT-IR peaks for the NH and C]O groups become
broad (Fig. 4). In the as synthesized compound, the bands at
3390 cmꢀ1 and 3295 cmꢀ1 indicate that some NH groups are free
and the rest are hydrogen bonded. The amide I and amide II
bands appeared at 1683, 1641 and 1526 cmꢀ1. From Fig. 4b, in
vesicles the amide I band at 1646 cmꢀ1 and amide II band at 1530
To investigate the topography of these microvesicles, atomic
force microscopy (AFM) studies were performed. The freshly
prepared solution of peptide 1 in methanol (0.5 mg mLꢀ1) was
drop-casted onto a microscopic glass coverslip and investigated
by AFM. Fig. 3a shows the AFM images of polydispersed
vesicles with diameters ranging from 250–500 nm. Fig. 3b shows
Fig. 2 FE-SEM images of mesoporous microvesicles of peptide 1 from
methanol at concentrations of: (a) 0.5 mg mLꢀ1, (b) 1 mg mLꢀ1 and (c)
Fig. 4 FT-IR spectra of (a) as synthesized peptide 1 and (b) peptide 1
and (d) 5 mg mLꢀ1
.
vesicles from methanol.
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Table 1 Selected backbone torsion angles (ꢁ) of peptide 1
cmꢀ1 suggest that the peptide adopts a kink like backbone
conformation.28
C4 N1 C5 C6
N1 C5 C6 N2
C6 N2 C7 C8
N2 C7 C8 N3
C8 N3 C9 C10
80.01
19.35
51.54
43.62
76.86
f1
j1
f2
j2
f3
N3 C9 C10 N4
11.45
104.17
14.94
j3
f4
j4
f5
j5
The kink like structure of the peptide 1 was further studied by
circular dichroism spectroscopy in methanol (ESI Fig. 3†) and
single crystal X-ray diffraction. The colorless hexagonal crystal
of the peptide 1 was obtained from a methanol–water solution by
slow evaporation.29 The molecular structure of peptide 1 with the
atomic numbering scheme is depicted in Fig. 5.
C10 N4 C11 C12
N4 C11 C12 N5
C12 N5 C101 C13
N5 C101 C13 O6
ꢀ52.09
ꢀ43.61
Most of the torsion angles (Table 1) of peptide 1 are in the left
hand helical region of the Ramachandran diagram (except f5 ¼
ꢀ52.09ꢁ and j5 ¼ ꢀ43.61ꢁ), resulting in a kink like conformation
for the individual peptide 1 molecule in the solid state. From
Fig. 5 there is only one ten member intramolecular hydrogen
shows that the vesicle surfaces are porous. We have tried the
encapsulation study with a potent bacteriostatic antibiotic sul-
famethoxazole (SMZ). The absorption and emission spectro-
scopic studies show that the microvesicles efficiently encapsulate
the drug. Fig. 8a shows a blue shift with increasing intensity upon
addition of peptide 1 solution in SMZ, indicating the formation
of a peptide 1 –drug complex. The fluorescence study (Fig. 8b)
also shows the gradual decrease of intensity with increasing
amount of peptide 1 to SMZ.
ꢁ
ꢀ
ꢀ
bond N13–H100/O12 (2.05 A, 2.87 A, 161 ). The individual
kink like sub-units of peptide 1 are themselves regularly inter-
linked through two intermolecular hydrogen bonding interac-
ꢁ
ꢁ
ꢀ
ꢀ
tions N10–H5/O15 (2.53 A, 3.03 A, 118 , x ꢀ y, x, ꢀ1/6 + z)
ꢀ
ꢀ
and N11–H8/O15 (2.26 A, 2.99 A, 143 , x ꢀ y, x, ꢀ1/6 + z)
thereby form a supramolecular helix along the crystallographic c
axis (Fig. 6a). The side view (Fig. 6a) and top view (Fig. 6b)
exhibit that the aromatic rings of the phenylalanine residues have
arranged in a spiral way around the supramolecular helical
column. In a higher order assembly, four supramolecular helical
columns are interdigitated through the noncovalent interaction
to form a supramolecular helical bundle structure (Fig. 6c and d).
Moreover, we have compared the data from wide angle X-ray
scattering of dried microvesicles and the powder pattern from X-
ray crystallography. The X-ray data indicates that the structures
of peptide 1 in microvesicles and as crystals are very close (ESI
Fig. 4†). Hence, we propose that the supramolecular helical
bundles are further self-assembled to form the microvesicles
(Fig. 7).
To investigate whether the drug molecules are encapsulated
inside the peptide microvesicles or adsorbed on the vesicle
surface, time resolved florescence studies of the bare drug and the
drug–peptide conjugate have been performed using the TCSPC
technique. The fluorescence decay curves of bare sulfamethox-
azole and sulfamethoxazole incorporated peptide spheres were
fitted with the equation:
X
n
IðtÞ ¼
Aiexpðꢀt=siÞ
i¼1
where Ai and si are the amplitude and lifetime of the ith flores-
cence component respectively and n is the number of fluorescence
exponentials required for the best nonlinear least squares
(NLLS) fitting of the fluorescence decay curves. When fitted with
the decay curve of bare SMZ, the single exponential curve with
a lifetime of 0.563 ns is obtained. But the decay curve for SMZ
encapsulated peptide spheres fitted well with the biexponential
decay curve (Fig. 9). The lifetimes were 0.425 ns with relative
amplitude 0.903 and 1.18 ns with a relative amplitude of 0.097
(Table 2). The decay curve for SMZ encapsulated peptide vesicles
shows that a second fluorescence component appeared with
Previous reports have shown that the peptide derived micro-
vesicles as drug carriers have many advantages including
biocompatibility, nontoxicity, adjustable controlled-release and
biodegradability.30 The FE-SEM study of peptide 1 microvesicles
Fig. 6 Supramolecular helical column of peptide 1 (a) side view; (b) top
view and interdigitated helical bundle of peptide 1 (c) side view and, (d)
top view.
Fig. 5 The molecular structure with atom numbering of peptide 1 in the
solid state. The intramolecular hydrogen bond is shown as a dotted line.
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encapsulation efficiency (%). To 3 mL of 3.94 ꢂ 10ꢀ2 mM drug
solution in methanol, 5 mg peptide 1 was added and stirred
overnight, the solvent was evaporated and the vesicles were
washed with 5 mL water. The residue was dissolved in 3 mL
methanol and absorption spectra were taken. The encapsulation
efficiency was calculated as (amount of drug added ꢀ amount of
free drug)/amount of drug added. The encapsulation efficiency of
peptide 1 vesicles is 32.20%, and drug loading content (i.e. (the
weight of the encapsulated drug in the vesicles/weight of vesicles
used) ꢂ 100) for these vesicles is 6.44%.
Further, to assess the encapsulation of sulfamethoxazole by
the microvesicles, DLS experiments were performed. The DLS
study exhibits that there is little change in particle size by loading
of the drug which indicates that the drug is encapsulated inside
the vesicles (ESI Fig. 5†). Moreover, the FE-SEM experiments
also show that the morphology of the microvesicle does not
change by drug encapsulation (Fig. 10). The existence of sulfur
by EDS studies further supports the encapsulation of sulfame-
thoxazole by the microvesicles (ESI Fig. 6†).
Fig. 7 A schematic presentation of microvesicle formed from supra-
molecular helical bundles of peptide 1.
We have also studied the release of the encapsulated sulfa-
methoxazole from peptide 1 microvesicles. 2 mg of the sulfa-
methoxazole loaded microvesicles were immersed into 5 mL
sodium phosphate buffer (pH 6.2) in a 15 mL centrifuge tube and
centrifuged at 5000 rpm for 5 min and monitored by UV-Visible
spectroscopy at different time intervals (ESI Fig. 7†). Under
slightly acidic conditions (sodium phosphate buffer of pH 6.2),
the protonation of sulfamethoxazole NH2 increases the hydro-
philicity of the drug molecules and governs the slow release from
the vesicles. Fig. 11 shows that the peptide microvesicles are
slowly releasing the encapsulated drug with a complete release by
24 h. At strongly acidic or basic pH, deprotection of the Boc and
–OMe groups affected the self-assembly of the peptide and
microvesicle structure (ESI Fig. 8†) and released the encapsu-
lated drug very fast.
Fig. 8 (a) Absorption spectra of sulfamethoxazole (3.94 ꢂ 10ꢀ2 mM)
with increasing peptide concentration. b) Emission of sulfamethoxazole
(3.94 ꢂ 10ꢀ2 mM) with increasing peptide concentration (lexcitation
¼
257nm).
Experimental
General
All L-amino acids were purchased from Sigma chemicals. HOBt
(1-hydroxybenzotriazole) and DCC (dicyclohexylcarbodiimide)
were purchased from SRL.
Fig. 9 Time dependent decay curves of sulfamethoxazole (SMZ) (green)
and peptide–drug conjugate (red). Black curve indicates instrumental
response function (IRF).
Peptide synthesis
The peptides were synthesized by a conventional solution-phase
method using a racemization free fragment condensation
Table 2 Nonlinear least squares (NLLS) fitting parameters obtained
from fluorescence decay curves (Fig. 9)
Sample
s1 (ns)
A1
s2 (ns)
A2
c2
SMZ
SMZ–vesicle
0.563
0.425
1
0.903
1.13
1.23
1.18
0.097
a lifetime of 1.18 ns due to complex formation of peptide vesicles
with drug molecules. The lifetime of the free drug molecules
decreased from 0.563 ns to 0.425 ns in the SMZ encapsulated
peptide vesicles due to collision quenching between peptide
molecules and free drug molecules in the solution.
The concentration of the drug molecules is quantified by
UV-Visible spectroscopy and the results are expressed as
Fig. 10 FE-SEM image of sulfamethoxazole encapsulated peptide 1
microvesicles.
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by 3.34 g (16.22 mmol) dicyclohexylcarbodiimide (DCC) and
2.48 g (16.22 mmol) of HOBt. The reaction mixture was allowed
to come to room temperature and stirred for 48 h. DCM was
evaporated and the residue was dissolved in ethyl acetate (60
mL) and dicyclohexylurea (DCU) was filtered off. The organic
layer was washed with 2 M HCl (3 ꢂ 50 mL), brine (2 ꢂ 50
mL), 1 M sodium carbonate (3 ꢂ 50 mL) and brine (2 ꢂ 50 mL)
and dried over anhydrous sodium sulfate; and evaporated in
a vacuum to yield Boc-Leu-Aib-OMe, 3, as a white solid. The
product was purified by silica gel (100–200 mesh) using n
hexane–ethyl acetate (3 : 1) as eluent. Yield: 4.17 g (12.62 mmol,
77.8%).
1H NMR (CDCl3, 500 MHz, dppm): 6.60 [s, 1H, Aib(2) NH],
4.78 [b, 1H, Leu(1) NH], 3.98 [b, 1H, Leu (1)Ca H], 3.66 [s, 3H,
OMe], 1.62–1.55 [m, 2H, Leu(1) CbH], 1.46 [s, 6H, Aib(2) CbH],
1.41 [s, 9H, BOC], 0.88–0.85 [m, 7H, Leu(1) CdH + CgH]. 13C
NMR (CDCl3, 125 MHz, dppm): 174.74, 171.73, 155.83, 80.07,
56.37, 56.37, 52.95, 52.53, 40.82, 28.26, 24.74, 24.70, 24.67, 22.86,
22.06. FT-IR (KBr): 3323, 3078, 2959, 2872, 1748, 1685, 1534,
1458, 1393, 1366, 1320, 1281, 1247, 1153 cmꢀ1. Anal calcd for
C16H30N2O5 (330.21): C, 58.16; H, 9.15; N, 8.48. Found: C,
58.20; H, 9.15; N, 8.50.
Fig. 11 Sulfamethoxazole release profile of peptide 1 microvesicles in
sodium phosphate buffer at pH 6.2.
strategy. The Boc group was used for N-terminal protection and
the C-terminus was protected as a methyl ester. Couplings were
mediated by dicyclohexylcarbodiimide/1-hydroxybenzotriazole
(DCC/HOBt). Methyl ester deprotection was performed via the
saponification method. All the intermediates were characterized
by 500 MHz 1H NMR and mass spectrometry. Final compounds
were fully characterized by 500 MHz 1H NMR spectroscopy, 13
C
NMR spectroscopy, mass spectrometry, and IR spectroscopy.
The peptide 1 was characterized by X-ray crystallography. The
products were purified by column chromatography using silica
(100–200 mesh size) gel as the stationary phase and n-hexane–
ethyl acetate mixture as eluent.
(c) Boc-Leu(1)-Aib(2)-OH (4). To 4.13g (12.5 mmol) of
compound 3, 25 mL MeOH and 2 M 15 mL NaOH were added
and the progress of saponification was monitored by thin layer
chromatography (TLC). The reaction mixture was stirred. After
10 h, methanol was removed under vacuum; the residue was
dissolved in 50 mL of water, and washed with diethyl ether (2 ꢂ
50 mL). Then the pH of the aqueous layer was adjusted to 2 using
1 M HCl and it was extracted with ethyl acetate (3 ꢂ 50 mL). The
extracts were pooled, dried over anhydrous sodium sulfate, and
evaporated under vacuum to obtained compound 4 as a white
solid. Yield 3.43 g (10.85 mmol, 86.8%).
1H NMR (DMSO-d6, 500 MHz, dppm): 12.20 [s, 1H, COOH],
7.92 [s, 1H, Aib(1) NH], 6.74–6.72 [d, 1H, J ¼ 9 Hz, Leu(1) NH],
3.98–3.34 [m, 1H, Leu(1) CaH], 1.63–1.56 [dd, 2H, Leu(1) CbH],
1.37 [s, 6H, Aib(2) CbH], 1.32 [s, 9H, Boc], 0.89–0.84 [m, 7H, Leu
(1) CdH + CgH].13C NMR (DMSO-d6, 125 MHz, dppm): 175.43,
171.69, 155.16, 77.91, 54.72, 52.44, 40.97, 28.14, 24.65, 24.12,
22.96, 21.60. FT-IR (KBr): 3308, 3067, 1665, 1528, 1376, 1284,
1171 cmꢀ1. Anal calcd. for C15H28N2O5 (316.20): C, 56.94; H,
8.92; N, 8.85. Found: C, 57.01; H, 8.90; N, 8.87.
(a) Boc-Leu(1)-OH (2). A solution of L-leucine (2.62 g, 20
mmol) in a mixture of dioxane (40 mL), water (20 mL) and 1 M
NaOH (20 mL) was stirred and cooled in an ice-water bath. Di-
tert-butylpyrocarbonate (4.8 g, 22 mmol) was added and stirring
was continued at room temperature for 6 h. Then the solution
was concentrated in vacuum to about 20–30 mL, cooled in an ice-
water bath, covered with a layer of ethyl acetate (about 50 mL)
and acidified with a dilute solution of KHSO4 to pH 2–3 (Congo
red). The aqueous phase was extracted with ethyl acetate and this
operation was done repeatedly. The ethyl acetate extracts were
pooled, washed with water and dried over anhydrous Na2SO4
and evaporated in a vacuum. The pure material was obtained as
a waxy solid. Yield: 3.80 g (16.4 mmol, 82.0%).
1H NMR (500 MHz, DMSO-d6, dppm): 12.36 [b, 1H, COOH],
7.03–7.02 [d, 1H, J ¼ 8 Hz, Leu(1) NH], 3.91–3.87 [m, 1H, Leu(1)
CaH], 1.63–1.60 [m, 2H, Leu(1) CbH], 1.51–1.47 [m, 1H, Leu(1)
CgH], 1.37 [s, 9H, BOC], 0.87–0.83 [m, 12H, 4 CH3]. 13C NMR
(125 MHz, DMSO-d6, dppm): 174.73, 155.64, 77.76, 66.41, 51.81,
28.24, 24.38, 22.29. FT-IR (KBr): 3462, 3355, 3311, 2983, 2968,
2934, 2877, 2525, 1727, 1699, 1675, 1533, 1456, 1419, 1385, 1368,
1296, 1197, 1172, 1087, 1048, 1018 cmꢀ1. Anal calcd for
C11H21NO4 (231.15): C, 57.12; H, 9.15; N, 6.06. Found: C, 57.15;
H, 9.12; N, 6.01.
(d) Boc-Leu(1)-Aib(2)-Phe(3)-OMe (5). 3.4 g (10.774 mmol)
Boc-Leu-Aib-OH was dissolved in 15 mL dry DCM in an ice-
water bath. 3.38 g (22 mmol) H-Phe-OMe was isolated from the
corresponding methyl ester hydrochloride by neutralization,
subsequent extraction with ethyl acetate and concentrated to 7
mL. Then it was added to the reaction mixture, followed
immediately by 2.222 g (10.774 mmol) dicyclohexylcarbodiimide
(DCC) and 1.649 g (10.774 mmol) HOBt. The reaction mixture
was allowed to come to room temperature and stirred for 72 h.
The residue was taken into 30 mL ethyl acetate and dicyclo-
hexylurea (DCU) was filtered off. The organic layer was washed
with 2 M HCL (3 ꢂ 50 mL), brine (2 ꢂ 50 mL), then 1 M sodium
carbonate (3 ꢂ 30 mL) and brine (2 ꢂ 30 mL) and dried over
anhydrous sodium sulfate and evaporated under vacuum to yield
the tripeptide 5 as a white solid. Purification was done by silica
(b) Boc-Leu(1)-Aib(2)-OMe (3). 3.75 g (16.22 mmol) of Boc-
Leu-OH was dissolved in 25 mL dry DCM in an ice-water bath.
H-Aib-OMe 4.915 g (32 mmol) was isolated from the corre-
sponding methyl ester hydrochloride by neutralization, subse-
quent extraction with ethyl acetate and solvent evaporation. It
was then added to the reaction mixture, followed immediately
10178 | Soft Matter, 2011, 7, 10174–10181
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gel column (100–200 mesh size) and ethyl acetate and hexane
(1 : 4) as eluent. Yield: 4.06 g (8.5 mmol, 78.89%).
1H NMR (CDCl3, 500 MHz, dppm): 7.39–7.37 [d, 1H, J ¼ 8.5,
Phe(3) NH], 7.25–7.11 [m, 10H, ring protons], 6.775–6.768 [d,
1H, J ¼ 7.5 Hz, Phe(4) NH], 6.55 [s, 1H, Aib(2) NH], 4.97–4.95
[d, 1H, J ¼ 7.5 Hz, Leu(1) NH], 4.78–4.77 [m, 1H, Phe(3) CaH],
4.68 [m, 1H, Phe(4) CaH], 3.66 [s, 3H, OMe], 3.88–3.86 [m, 1H,
Leu(1) CaH], 3.30–2.89 [dm, 2H, Phe 2 CbH], 1.60–1.56 [m, 2H,
Leu(1) CbH], 1.42 [s, 15 H, CbH Aib(2) + Boc], 0.94–0.89 [m, 7H,
Leu(1) CdH + CgH]. 13C NMR (CDCl3, 125 MHz, dppm): 173.36,
172.64, 171.87, 170.98, 137.35, 136.93, 129.25, 128.93, 128.67,
128.59, 128.45, 128.38, 126.71, 126.64, 80.96, 56.85, 54.05, 53.73,
52.18, 40.05, 37.60, 36.82, 28.27, 25.81, 24.96, 24.76, 24.51, 22.95,
21.83. FT-IR (KBr): 3309, 3063, 3029, 2956, 2866, 1733, 1664,
1527, 1366, 1366, 1254, 1219, 1168 cmꢀ1. Anal calcd. for
C34H48N4O7 (624.35): C, 65.36; H, 7.74; N, 8.97. Found: C,
65.38; H, 7.76; N, 8.93.
1H NMR (CDCl3, 500 MHz, dppm): 7.29–7.11 [m, 5H,
aromatic protons], 6.85 [b, 1H, Phe(3) NH], 6.62 [s, 1H, Aib(2)
NH], 4.82–4.79 [m, 1H, Phe(3) CaH], 3.98 [b, 1H, Leu(1) CaH],
3.70 [s, 3H, OMe], 3.15–3.08 [m, 2H, CbH Phe], 1.64–1.60 [m, 2H,
Leu(1) CbH], 1.48 [s, 6H, Aib(2) CbH], 1.44 [s, 9H, Boc], 0.94–
0.91 [m, 7H, Leu(1) CdH + CgH]. 13C NMR (CDCl3, 125 MHz,
dppm): 173.78, 171.91, 129.34, 129.25, 128.61, 128.49, 127.02,
57.18, 53.44, 52.26, 37.82, 28.28, 25.20, 25.02, 24.95, 24.72, 23.00,
21.92. FT-IR (KBr): 3442, 3406, 3336, 2956, 2933, 1741, 1708,
1673, 1508, 1458, 1364, 1279, 1167 cmꢀ1. Anal calcd. for
C25H39N3O6 (477.28): C, 62.87; H, 8.23; N, 8.80. Found: C,
62.85; H, 8.25; N, 8.76.
(e) Boc-Leu(1)-Aib(2)-Phe(3)-OH (6). To 4.0 g (8.37 mmol)
of compound 5, 25 mL MeOH and 2 M 15 mL NaOH were
added and the progress of saponification was monitored by
thin layer chromatography (TLC). The reaction mixture was
stirred. After 10 h, methanol was removed under vacuum; the
residue was dissolved in 50 mL of water, and washed with
diethyl ether (2 ꢂ 50 mL). Then the pH of the aqueous layer
was adjusted to 2 using 1 M HCl and it was extracted with
ethyl acetate (3 ꢂ 50 mL). The extracts were pooled, dried over
anhydrous sodium sulfate, and evaporated under vacuum to
obtain the compound as a waxy solid. Yield: 3.67 g (7.93 mmol,
94.74%).
1H NMR (DMSO-d6, 500 MHz, dppm):12.56 [b, 1H, COOH],
7.88 [s, 1H, Aib(2) NH], 7.50–7.48 [d, 1H, J ¼ 7.5 Hz, Phe(3)
NH], 7.275–7.167 [m, 5H, ring protons], 6.94–6.93 [d, 1H,J ¼ 7.5
Hz, Leu(1) NH], 4.40–4.39 [m, 1H, Phe(3) CaH], 3.88–3.87 [m,
1H, Leu(1) CaH], 3.06–2.90 [dm, 2H, Phe(3) CbH], 1.57–1.55 [m,
2H, Leu(1) CbH], 1.35 [s, 6H, Aib(2) CbH], 1.33 [s, 9H, Boc],
0.88–0.82 [m, 7H, Leu(1) CdH + CgH]. 13C NMR (DMSO-d6, 125
MHz, dppm): 173.74, 172.61, 171.99, 155.46, 137.42, 129.16,
129.01, 128.14, 128.04, 126.33, 78.06, 55.84, 53.53, 53.05, 36.73,
28.14, 24.92, 24.18, 4.05, 23.00, 21.49. FT-IR (KBr): 3413, 3315,
2955, 2928, 2870, 1664, 1526, 1371, 1288, 1172 cmꢀ1. Anal calcd.
for C24H37N3O6 (463.27): C, 62.18; H, 8.04; N, 9.06. Found: C,
62.15; H, 8.08; N, 9.01.
(g) Boc-Leu(1)-Aib(2)-Phe(3)-Phe(4)-OH (8). To 3.3 g (5.28
mmol) of compound 6, 25 mL MeOH and 2 M (15 mL) NaOH
were added and the progress of saponification was monitored by
thin layer chromatography (TLC). The reaction mixture was
stirred. After 10 h, methanol was removed under vacuum; the
residue was dissolved in 50 mL of water, and washed with diethyl
ether (2 ꢂ 50 mL). Then the pH of the aqueous layer was adjusted
to 2 using 1 M HCl and it was extracted with ethyl acetate (3 ꢂ 50
mL). The extracts were pooled, dried over anhydrous sodium
sulfate, and evaporated under vacuum to obtain the compound as
a waxy solid. Yield 2.74 g (4.49 mmol, 85.0%).
1H NMR (DMSO-d6, 500 MHz, dppm):12.67 [s, 1H, COOH],
8.08–8.06 [d, 1H, J ¼ 8.0 Hz, Phe(3) NH], 7.94 [s, 1H, Aib(2) NH],
7.45–7.43 [d, 1H, J ¼ 8.5 Hz, Phe(4) NH], 7.28–7.14 [m, 10H, ring
protons], 6.93–6.92 [d, 1H, 7.5 Hz, NH Leu], 4.44–4.38 [m, 2H,
Phe 2CaH], 3.90–3.78 [m, 1H, Leu(1) CaH], 3.11–2.79 [ddm, 4H, 2
CbH Phe], 1.63–1.53 [m, 2H, Leu(1) CbH], 1.36 [s, 9 H, Boc],1.23 [s,
6H, Aib(2) CbH], 0.85–0.80 [m, 7H, Leu(1) CdH + CgH]. 13C NMR
(DMSO-d6, 125 MHz, dppm): 173.41, 172.56, 172.48, 170.76,
155.60, 137.92, 137.58, 129.05, 128.15, 127.89, 126.37, 126.09,
78.25, 55.84, 53.77, 53.68, 53.26, 36.68, 36.54, 28.13, 24.54, 24.13,
22.89, 21.54. FT-IR (KBr): 3309, 2955, 1655, 1528, 1450, 1367,
1168, 1019 cmꢀ1 Anal calcd. for C33H46N4O7 (610.33): C, 64.90;
H, 7.59; N, 9.17. Found: C, 64.96; H, 7.62; N, 9.15.
(f) Boc-Leu(1)-Aib(2)-Phe(3)-Phe(4)-OMe (7). 3.5 g (7.55
mmol) of compound 5 was dissolved in 25 mL dry DCM in an
ice-water bath. H-Phe-Ome was isolated from 3.45 g (16 mmol)
of the corresponding methyl ester hydrochloride by neutraliza-
tion, subsequent extraction with ethyl acetate and solvent evap-
oration. It was then added to the reaction mixture, followed
immediately by the addition of 1.557 g (7.55 mmol) dicyclohexy-
lcarbodiimide (DCC) and 1.156 g (7.55 mmol) of HOBt. The
reaction mixture was allowed to come to room temperature and
stirred for 72 h. DCM was evaporated and the residue was dis-
solved in ethyl acetate (60 mL) and dicyclohexylurea (DCU) was
filtered off. The organic layer was washed with 2 M HCl (3 ꢂ 50
mL), brine (2 ꢂ 50 mL), 1 M sodium carbonate (3 ꢂ 50 mL) and
brine (2 ꢂ 50 mL) and dried over anhydrous sodium sulfate; and
evaporated in a vacuum to yield compound 7. as a white solid.
The product was purified by column chromatography by 100–
200 mesh silica gel using hexane–ethyl acetate (6 : 1) as eluent.
Yield 3.4 g (5.45 mmol, 72.3%).
(h) Boc-Leu-Aib-Phe-Phe-Aib-OMe (1). 2.65 g (4.33 mmol)
of compound 7 were dissolved in 25 mL dry DCM in an ice-water
bath. H-Aib-OMe was isolated from 1.38 g (9.0 mmol) of the
corresponding methyl ester hydrochloride by neutralization,
subsequent extraction with ethyl acetate and solvent evapora-
tion. It was then added to the reaction mixture, followed
immediately by the addition of 0.893 g (4.33 mmol) dicyclohexy-
lcarbodiimide (DCC) and 0.663 g (4.33 mmol) of HOBt. The
reaction mixture was allowed to come to room temperature and
stirred for 72 h. DCM was evaporated and the residue was dis-
solved in ethyl acetate (60 mL) and dicyclohexylurea (DCU) was
filtered off. The organic layer was washed with 2 M HCl (3 ꢂ 50
mL), brine (2 ꢂ 50 mL), 1 M sodium carbonate (3 ꢂ 50 mL) and
brine (2 ꢂ 50 mL) and dried over anhydrous sodium sulfate;
and evaporated in a vacuum to yield Boc-Leu-Val-Aib-Phe-Aib-
OMe as a white solid. The product was purified by column
chromatography by 100–200 mesh silica gel using hexane–ethyl
acetate (3 : 1) as eluent. Yield 2.62 g (3.59 mmol, 82.9%).
This journal is ª The Royal Society of Chemistry 2011
Soft Matter, 2011, 7, 10174–10181 | 10179

View Article Online
1H NMR (CDCl3, 500 MHz, dppm): 7.80–7.78 [d, 1H, J ¼ 9.0
Hz, Phe (3) NH], 6.80 [s, 1H, Aib NH 1], 6.760 [s, 1H, NH Aib 2],
6.771–6.760 [d, 1H, 5.5 Hz, NH Phe 2], 5.497 [b, 1H, NH Leu],
4.681–4.657 [m, 1H, CaH Phe 1], 4.440–4.420 [m, 1H, CaH Phe 2],
3.949–3.944 [m, 1H, CaH Leu], 3.486–3.452, 3.114–3.074, 3.000–
2.948, 2.744–2.714 [ddm, 4H, 2 CbH Phe], 1.544, 1.496 [s, 6H,
Aib1], 1.432 [b, 2H, CbH Leu], 1.377 [s, 9H, Boc], 1.248, 1.208 [s,
6H, Aib 2], 0.960–0.911 [m, 7H, CdH + CgH Leu]. 13C NMR
(CDCl3, 125 MHz, dppm): 175.16, 174.90, 173.34, 171.09, 170.78,
156.77, 138.56, 136.76, 129.19, 128.54, 128.35, 128.20, 126.68,
126.27, 80.95, 56.31, 55.96, 55.72, 54.13, 52.27, 39.56, 36.90, 36.51,
36.14, 29.64, 28.14, 26.20, 25.34, 24.72, 24.55, 23.98, 22.81, 14.07.
FT-IR (KBr): 3391, 3292, 3031, 2960, 2870, 1748, 1684, 1642,
1525, 1457, 1386, 1365, 1302, 1275, 1249, 1161 cmꢀ1. Mass spectra:
[M + H]+: 710.4457 (actual: 710.4051). [M + Na]+: 732.4025
(actual: 732.3948). Anal calcd. for C38H55N5O8 (709.40): C, 64.29;
H, 7.81; N, 9.87. Found: C, 64.30; H, 7.84; N, 9.85.
Fluorescent spectroscopy
Fluorescent spectra were recorded on a fluorescent spectrometer
(Perkin Elmer) in methanol at excitation wavelength 257 nm.
Concentration of sulfamethoxazole was 3.94 ꢂ 10ꢀ2 mM and
peptide concentration was 10 mM.
Time resolved fluorescence spectroscopy
The fluorescence lifetimes were measured by the method of time-
correlated single photon counting using a picosecond spectro-
fluorometer from Horiba Jobin Yvon IBH. The instrument was
equipped with FluoroHub single photon counting controller,
Fluoro3PS precision photomultiplier power supply, and FC-
MCP-50SC MCP-PMT detection unit. A laser head or a nano-
LED pulsed diode powered by a pulsed diode controller (IBH)
was used as the excitation light source. The excitation was done
at 340 nm wavelength. The sulfamethoxazole was taken at
a concentration of 4 ꢂ 10ꢀ2 mM in methanol. For the drug–
peptide conjugate, 100 mL 10 mM peptide solution were added to
2.9 mL 4 ꢂ 10ꢀ2 mM drug solution. The typical response time of
this laser head was <100 ps. To calculate the lifetime, the fluo-
rescence decay curves were analyzed by an iterative fitting
program provided by IBH.
NMR experiments
€
All NMR studies were carried out on a Bruker AVANCE 500
MHz spectrometer at 278 K. Compound concentrations were in
the range of 1–10 mM in CDCl3 and (CD3)2SO.
FTIR spectroscopy
Dynamic light scattering
All reported solid-state FTIR spectra were obtained using
a Perkin Elmer Spectrum RX1 spectrophotometer with the KBr
disk technique.
The particles sizes of the nanovesicles were determined by DLS
instrument (model ZETASIZER nano series nano zs) with the
peptide concentrations 0.5 mg mLꢀ1, 1 mg mLꢀ1 and 2 mg mLꢀ1
in methanol. For drug loaded vesicles, 1 mg mLꢀ1 of drug loaded
vesicles were dissolved in methanol and studied.
Mass spectrometry
Mass spectra were recorded on a Q-Tof Micro YA263 high-
resolution (Waters Corporation) mass spectrometer by positive-
mode electrospray ionization.
Encapsulation procedure
20 mg of peptide 1 and 1 mg drug were dissolved in 5 mL
methanol and stirred overnight. The solution was drop casted on
a Petri dish and dried. Finally, the drug loaded vesicles were
dried under vacuum and washed with double distilled water
(several times) to remove non encapsulated drugs.
Scanning electron microscopy
The morphologies of the reported materials were investigated by
field emission scanning electron microscope (FESEM). For the
SEM study, the methanol solutions of peptide 1 at concentra-
tions of 0.5 mg mLꢀ1, 1 mg mLꢀ1 and 5 mg mLꢀ1 were drop
casted onto microscopic glass slides, dried and coated with
platinum. Then the micrographs were taken in a SEM apparatus
(JEOL microscope JSM-6700F).
X-ray crystallography
Single crystal X-ray analysis of peptide 1 was recorded on
a Bruker high resolution X-ray diffractometer instrument.
Wide angle X-ray diffraction study
Atomic force microscopy
The experiment was carried out in a Seifert X-ray diffractometer
(C 3000) with a parallel beam optics attachment. The instrument
was operated at a 45 KV voltage and 200 mA current and was
calibrated with a standard silicon sample. The dried peptide 1
microvesicles from methanol were scanned from 5ꢁ to 45ꢁ (2q) at
the step scan mode (step size 0.02ꢁ, preset time 2 s) and the
diffractionpatternwasrecordedusinga scintillationscandetector.
The wave length of the X-ray source is 1.5418 A0 (Cu-Ka).
The morphologies of the reported peptide were investigated by
AFM. A small amount of solution (0.5 mg mLꢀ1 MeOH) of the
corresponding compounds was placed on a microscope cover
glass and then dried by slow evaporation. The material was then
ꢁ
allowed to dry in a vacuum at 30 C for two days. Images were
taken with an NTMDT instrument, model no. AP-0100 in
semicontact-mode.
UV/Vis spectroscopy
Conclusions
UV/Vis absorption spectra were recorded on a UV/Vis spectro-
photometer (Hitachi). Concentration of sulfamethoxazole was
3.94 ꢂ 10ꢀ2 mM and peptide concentration was 10 mM.
In conclusion, fabrication of mesoporous microvesicles from
a self-assembled hydrophobic peptide has been discussed. The
10180 | Soft Matter, 2011, 7, 10174–10181
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15 S. A. Hagan, A. G. A. Coombes, M. C. Garnett, S. E. Dunn,
M. C. Davies, L. Illum, S. S. Davis, S. E. Harding, S. Purkiss and
P. R. Gellert, Langmuir, 1996, 12, 2153.
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10007.
peptide forms a supramolecular helical column through intra-
and intermolecular hydrogen bonding interactions and inter-
digitated helical bundle structures in the solid state. The peptide
forms mesoporous microvesicles in methanol solution, where
diameters of the vesicles vary with the concentration in direct
proportion. Moreover, the mesoporous vesicular structures
encapsulate a potent bacteriostatic antibiotic sulfamethoxazole
and slowly release the encapsulated drug at pH 6.2, which might
be attractive in the field of drug delivery and responsive release.
€
€
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We acknowledge the DST, New Delhi, India, for financial
assistance, Project No. (SR/FT/CS-041/2009). S. Maity, P. Jana
and S. K. Maity acknowledge C.S.I.R., New Delhi, India for
financial assistance. We gratefully acknowledge the I.A.C.S.,
Jadavpur, Kolkata-700032, India for use of the FE-SEM facility.
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ꢀ
space group P65, a ¼ 10.974(8), b ¼ 10.974(8), c ¼ 59.527(7) A, U
3
¼ 6208 A , Z ¼ 6, dm ¼ 1.139 Mgmꢀ3 Intensity data were collected
ꢀ
with Mo-Ka radiation at room temperature using a Bruker APEX-
2 CCD diffractometer. 100 frames were measured at 2ꢁ intervals
with a counting time of 5 min to give 19943 independent reflections.
Data were processed using the Bruker SAINT package and the
structure solution and refinement procedures were performed using
SHELX97.31 The non-hydrogen atoms were refined with anisotropic
thermal parameters. The hydrogen atoms were included in
geometric positions and given thermal parameters equivalent to 1.2
times those of the atom to which they were attached. The final R
values were R1: 0.0762 and R2: 0.2449 for 3686 data with I > 2s(I).
The largest peak and hole in the final difference Fourier were ꢀ0.16
ꢀ3
ꢀ
and 0.27 e A
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Soft Matter, 2011, 7, 10174–10181 | 10181