Study of the nano-morphological versatility by self-assembly of a peptide mimetic molecule in response to physical and chemical stimuli

Article abstract of DOI:10.1039/c3cc49769k

A small peptide mimetic molecule can form diverse nanostructures such as nano-vesicles, nano-tubes and nano-ribbons/fibrils by self-assembly, in response to various physical and chemical stimulations. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3cc49769k

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Study of the nano-morphological versatility by
self-assembly of a peptide mimetic molecule in
response to physical and chemical stimuli†
Cite this: Chem. Commun., 2014,
50, 2638
Received 25th December 2013,
Accepted 10th January 2014
Sudeshna Kar,a Kung-Wei Wu,a I-Jui Hsu,b Chi-Rung Leec and Yian Tai*a
DOI: 10.1039/c3cc49769k
www.rsc.org/chemcomm
A small peptide mimetic molecule can form diverse nanostructures self-assembles to generate various nano-structures such as nano-
such as nano-vesicles, nano-tubes and nano-ribbons/fibrils by self- vesicles, nanotubes and nano-ribbons/fibers depending upon
assembly, in response to various physical and chemical stimulations. the solvent effect, substrate functionality and thermal treatment.
Such morphological diversities make it an attractive building block
Peptides have long been recognized as an important class of for model study and possible applications in nanobiotechnology.
molecules in biochemistry, medicinal chemistry, physiology, Moreover, this salt responsive PMM based vesicular structure has
and bionanotechnology.1–3 At a fundamental level, proteins are high efficiency of encapsulation, indicating the possibility of its
the combination of several small peptide fragments. Therefore use in model study for carrying drugs. The morphological diversity
an understanding of how small peptides respond to physical of the self-assembled nanostructures of the PMM was examined by
and chemical stimuli is very important for understanding various microscopic techniques. Field emission scanning electron
protein interaction behaviour in biological systems. Although microscopy (FE-SEM) reveals that the methanolic solution of the
morphological changes of small peptides, by varying self- PMM generates highly dense, well-structured, spherical objects
assembling parameters, for model study of various biological having size distribution with diameters of approximately 500 nm
phenomenons, have been studied by many groups in the last (Fig. 1(a) and (b)). The chemical structure of the PMM is demon-
few decades,4–9 e.g. Zhang et al. have studied the self-assembly strated in the inset of Fig. 1(f).
of surfactant-like peptides with variable glycine tails which
The number density and the diameter of the spheres vary
form nanotubes and nanovesicles in neutral aqueous solution,4 with respect to the PMM concentrations in methanolic solution
yet the behaviours such as surface-induced morphological (Fig. S1 in ESI†). The porous nature of vesicles is also clear from
transformation of peptide-based nanostructures and their solvent the SEM image (Fig. S2 in ESI†). Several larger spherical aggregates
induced inter-conversion have not been focused. In addition, were observed which formed through the fusion of smaller ones
study of morphological diversities of very small peptide mimetic (Fig. S3 in ESI†). The driving forces behind the fusion process may
molecules (PMMs), constituting only un-natural amino acids, has be the release of strain in the initially formed vesicles, which have
not been noticed yet.
Herein, we synthesized a new PMM, Boc-m-ABA-Aib-OMe
(m-ABA: m-aminobenzoic acid, Aib: a-aminoisobutyric acid), and
investigated the morphological transformations of this peptide-
based nanostructure under various stimulations. Interestingly
this newly synthesized terminally protected dipeptide molecule
a Department of Chemical Engineering, National Taiwan University of Science and
Technology, 43 Keelung Road, Taipei 10607, Taiwan.
E-mail: ytai@mail.ntust.edu.tw
b Department of Molecular Science and Engineering, National Taipei University of
Technology, Taipei 10608, Taiwan
c Department of Chemistry and Materials Engineering, Minghsin University of
Science and Technology, Hsin-chu 304, Taiwan
Fig. 1 FE-SEM image of vesicles generated by PMM (a), TEM image showing
the hollow nature of vesicles generated by PMM (b), one enlarged TEM image
of a vesicle has been shown in the inset of (b); FE-SEM image of PMM after
thermal treatment for 1 h at a constant temperature of (c) 50 1C, (d) 100 1C,
and (e) 150 1C, and (f) the chemical structure and the TGA plot of PMM.
† Electronic supplementary information (ESI) available: Materials, methods,
additional characterization, and crystallographic data. CCDC 949965. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c3cc49769k
2638 | Chem. Commun., 2014, 50, 2638--2641
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a high degree of curvature. By fusion into larger vesicles, the observed on both –CH3 and –C8 SAMs (Fig. S8(c) and (d), ESI†).
curvature energy decreases, leading to a thermodynamically Thus, we commented that the functionality on the surface, not
more stable state.10 Transmission electron microscopic (TEM) the chain length, has a great effect on the self-assembly pattern
studies were performed to investigate the internal structure of of peptides. It was previously proved by DLS study that in
these self-assembled spherical objects. It is found that the methanol medium the peptide forms nano-vesicles. Therefore
spheres obtained from the methanolic solution of the peptide we can assume that the self-assembly pattern of the peptide is
have hollow spaces inside, indicative of the vesicle configura- not disturbed when the methanolic solution of it is dropped
tions (Fig. 1(b) and Fig. S4 in ESI†). The dynamic light scattering on –NH2 or –SH SAMs. Because in methanol, the vesicles are
(DLS) experiment was performed in the methanolic solution of surrounded by the polar hydroxyl (–OH) group of methanol and
the PMM in order to assess the formation of self-assembled while the vesicles are dropped on –NH2 or –SH SAMs, the outer
structures in solution. The result showed that the nanostructures environment around the vesicles does not change to a great
display hydrodynamic diameter centered at 320.25 nm with extent. Therefore the vesicular structures remain unaltered.
polydispersity index, 0.30 (Fig. S5 in ESI†). The hydrodiameter However, when methanolic solution of the peptide is dropped
values that are higher than the average diameters observed in on different –CH3 SAM surfaces, having different chain lengths
TEM indicate extensive solvation of the nanoscopic structures or of alkyl silanes, the outer environment around the vesicles was
fusion of smaller vesicles to larger ones in the solution phase.
drastically changed. The PMM molecules tend to interact with
To examine the thermal effect on the nano-vesicles, we –CH3 functional groups in a way that causes an alteration in the
performed a comparative study by aging them in a convection higher order self-assembled packing of the peptide molecules.
oven at a constant temperature of 50, 100 and 150 1C for 1 h This experiment may provide insights for understanding the
each. The SEM and TEM results indicate that a morphological interaction of peptide/protein with different functional groups
change occurred from vesicular structures to fibrils and to plates at biological interfaces. Moreover the solvent induced inter-
(Fig. 1(c)–(e)) due to rise of temperature. Such morphological conversion of these nano-fibers to nano-vesicles in the presence
changes due to temperature variation might serve as a scaffold of methanol was also investigated (Fig. S9, ESI†). This experi-
for construction of various nano-devices where different process ment supports the fact that the influence of functional groups
temperatures are required.11 A complete destruction of all the present in solvent or on substrates are very important for the
nanostructures was observed when they were heated above higher order self-assembly pattern of PMM. This interconversion
150 1C (Fig. S6, ESI†), which can be further confirmed by the of nano-structures may facilitate biological, therapeutic and
thermogravimetric analysis (TGA) result (Fig. 1(f)).
cosmetic applications and may have a great impact for several
To understand the precise role of solvents in generating nano-technological applications.
different morphologies of the new peptide, solvent dependency
To investigate the secondary structure in self-assembled
experiments were carried out.5,6,12 We discovered that the PMM peptide vesicles, solid state FT-IR spectroscopy was performed.
forms elongated nano-tubular morphologies with a diameter of The important absorption bands of the vesicles have been com-
B200 nm and having several micrometer length from chloroform– pared with the as-synthesized compound (Table S1 and Fig. S10,
methanol (1 : 1 v/v) solvent medium (Fig. S7(a), ESI†), whereas ESI†). No peak at around 3430–3440 cmÀ1 was observed indicating
from toluene, the peptide forms short nanotubes (Fig. S7(b), ESI†). that all the –NH groups are involved in intermolecular hydrogen
In the presence of ethyl-acetate, well aligned nano-fibrils were bonding.14 The peaks at around 1745.73 cmÀ1 and 1745.56 cmÀ1
found to be generated (Fig. S7(c), ESI†). Therefore our PMM could for the as synthesized compound and the vesicles, respectively,
offer a model for the investigation of peptide fibrillation.8 On the indicate the CQO stretching vibrations of saturated non-H-
other hand, the PMM forms flat tape or ribbon like structure bonded methyl ester. From the position of the amide I band at
when grown from CHCl3–petroleum ether (1 : 1 v/v) solvent around 1644 cmÀ1 and the amide II band at around 1558 cmÀ1
,
medium, acetone and dimethylformamide solvents, individually it is assumed that the PMM forms b-sheet like structure in the
(Fig. S7(d)–(f), ESI†). From the results, it is found that there is a solid state.15–18 The IR result also reveals that the supramolecular
clear-cut impact of different solvent systems on the fabrication of b-sheet like structure is retained while forming fibrils on –CH3
morphological diversity, which may be due to the influence of SAM or generated from different solvents or after thermal treat-
different functional groups present in each solvent on the ment and during formation of tubes from aromatic solvent.
various noncovalent interactions responsible for self-assembly
of the peptide molecule.5
Single-crystal X-ray diffraction (XRD) results (Fig. S11 and
Tables S2–S4, ESI†) revealed that the PMM forms b-sheet like
Furthermore, we demonstrated the influence of surface layers through molecular self-assembly (Fig. 2(a)), in which the
functionality13 on the self-assembly of the PMM using self- m-ABA mediated aromatic p-staking interactions play an important
assembled monolayers (SAMs) generated on the glass surface role (Fig. S12, ESI†). Various noncovalent interactions such as
as a model system. We utilized four different SAMs, namely, intermolecular hydrogen bonding and hydrophobic interactions
3-aminopropyltriethoxysilane (–NH2), 3-mercaptopropyltri- play the key roles in the formation of the b-sheet layer like
ethoxysilane (–SH), propyltriethoxysilane (–CH3) and octyl- structure. These structures take part in higher order self-assembly
triethoxysilane (–C8). On both –NH2 and –SH SAMs, the PMM through non-specific van der Waals force and hydrophobic inter-
showed the formation of vesicles (Fig. S8(a) and (b), ESI†). actions (Fig. 2(b)). From the wide angle X-ray diffraction (WAXD)
However, formation of nano-ribbons/fibrils of the PMM was analysis (Fig. S13 and Table S5, ESI†) it is supposed that a similar
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Fig. 3 TEM image of (a) methanolic solution of 0.014 mM methotrexate
solution and (b) methotrexate loaded PMM vesicles after incubation for 2 days;
fluorescence microscopic images showing (c) green fluorescence of the
methotrexate loaded PMM vesicles after incubation for 2 days and (d) rupture
of methotrexate loaded PMM vesicles after 24 h incubation with 10 mM KCl
salt solution; (e) fluorescence emission spectra showing encapsulation and
release of the methotrexate drug at an excitation wavelength of 350 nm.
Fig. 2 Single crystal XRD results of (a) supramolecular b-sheet-like struc-
ture of PMM in the ab plane; (b) higher order self-assembly of the b-sheet
layers in the b-direction. Each sheet-like layer is indicated by a double
headed arrow, and (c) proposed schematic model of formation of various
nano-structures.
solution with 10 mM KCl solution (1 : 1 v/v mixing) for 24 h,
the fluorescence of MTX again increases, confirming effective
drug-release. In Fig. S15 (ESI†), we tried to provide a probable
mechanism of MTX drug penetration through the nano-pores
on the surface of the hollow PMM vesicles. Interestingly, the
vesicles can entrap the drug in its cavity for several days, even
up to 2 months (Fig. S16, ESI†), which indicates the stability of
self-assembly of the nano-vesicles. Notably, the morphological
pattern of the day or month aged MTX drug directly reflects the
morphology of the drug inside the peptide-vesicle (Fig. S16,
ESI†), depicting that there is no chemical interaction of the
PMM with the drug due to protection of the peptide molecule at
both ends and thereby highly recommending it to be utilized as
a drug carrier in therapeutics.
In summary, we have demonstrated the self-assembly of a
new di-peptide mimetic molecule, constituting only un-natural
amino acids, into various nanostructures under various stimu-
lations and its possible application in drug delivery. This newly
described small peptide mimetic molecule not only is attractive
for the model study of peptide interaction behaviours in bio-
logical systems, but also offers novel scaffolds for the future
design of new functional biomaterials.
type of crystal packing is responsible for the formation of
vesicle, tube and fibril/ribbon-like structures.8,19 The formation
of nano-vesicles may be envisaged by considering the wrapping
of the b-sheet-like layers in two different directions simulta-
neously20 as depicted in the schematic model in Fig. 2(c). We
assume that due to thermal treatment or interaction with –CH3
functional groups on different SAM surfaces or the presence of
different solvents, the two-way wrapping of b-sheet layers opens
up and they are arranged side by side to form the fibrils/ribbons
(Fig. 2(c)). Again in a chloroform–methanol solvent mixture (1 : 1 v/v)
and aromatic solvent like toluene, b-sheet-like layers may fold
in only one direction to form the nano-tubes (Fig. 2(c)).8,20
A simple co-relation with the crystal structure and the TEM
images of PMM nanostructures has been provided in Fig. S14 of
ESI,† which also supports our assumption.21
Thus, the self-assembly of m-amino benzoic acid and
a-amino isobutyric acid mediated peptide molecules, without
any electrostatic interactions, supports the fact that various
non-covalent interactions can facilitate the formation of such
well-ordered nano-morphologies.
We thank Dr S. G. Shyu (Academia Sinica) for the support
of XRD.
The design and construction of nano-vesicles from the self-
assembling peptide and pseudo peptides are being considered
as excellent vehicles for encapsulating and carrying drugs,22,23
but the entrapment capability of stable nano-vesicles generated
from small peptide-mimetic molecules is yet to be explored.
Thus, we investigated the efficiency of the PMM based nano-
vesicles to encapsulate the anti-cancer drug Methotrexate
(MTX).24 TEM images of the drug and drug after encapsulation
by the nano-vesicles clearly depict the encapsulation of the
MTX drug by the nano-vesicles formed by PMM (Fig. 3(a)
and (b)). Moreover, the entrapped drug can be released easily
by simple addition of a biocompatible metal salt, such as KCl,
into the drug-loaded vesicles, which was confirmed by fluores-
cence microscopic images (Fig. 3(c) and (d)). This encapsulation–
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