Cyclic peptide facial amphiphile preprogrammed to self-assemble into bioactive peptide capsules

Article abstract of DOI:10.1002/chem.200903145

(Figure Presented) Programmed assembly: A unique supramolecular building block, a cyclic peptide facial amphiphile, predestined to self-assemble into a nanocapsule structure, has been developed (see figure). This general strategy will allow the easy construction of a variety of bioactive nanocapsules.

Full text of DOI:10.1002/chem.200903145

COMMUNICATION
DOI: 10.1002/chem.200903145
Cyclic Peptide Facial Amphiphile Preprogrammed to Self-Assemble
into Bioactive Peptide Capsules
Eun Kyung Chung,[a] Eunji Lee,[a] Yong-beom Lim,*[b] and Myongsoo Lee*[a]
Self-assembled peptide nanostructures have shown great
potential as promising biomaterials.[1–8] Peptides have the
advantage of being intrinsically biocompatible materials.
Peptides, as self-assembling building blocks, have mostly
been designed to have head/tail molecular configuration.
Since there is a great deal of interest in the precise control
of a self-assembly process, novel building blocks significantly
different from those with conventional head/tail configura-
tion might provide ample opportunities for constructing
elaborate, versatile, and smart nanostructures.
Cyclic peptides, due to their constrained nature, are one
of those self-assembly building blocks that have unique
topological features compared with conventional linear
(head/tail) peptides.[9–11] Another important self-assembling
building block that has a special architecture is the facial
amphiphiles.[12–15] In facial amphiphiles, the hydrophilic and
-phobic groups are located on two opposite faces, rather
than at two ends as in head/tail amphiphiles. Herein, we
present an approach to construct a novel type of self-assem-
bling building blocks in which the characteristics of cyclic
peptides and facial amphiphiles are combined. The self-as-
sembly behavior of this novel cyclic peptide facial amphi-
phile (CPFA) could be regulated through the judicious
design of molecular structure, showing the power of this ra-
tional approach for precise nanostructural control.
of bioapplications, including drug, gene, and protein deliv-
ery.[16–21] Capsules have hollow interior within which cargo
molecules can be entrapped. Upon suitable functionalization
of outer shell, capsules can be made to bind or enter the
cells. Traditionally, self-assembled capsules have been fabri-
cated by using amphiphilic molecules of head/tail configura-
tion, such as amphiphilic lipids and block copolymers. The
supramolecular morphology of the head/tail amphiphiles de-
pends highly on the relative volume fraction between hydro-
philic and -phobic blocks.[19,22–24] Therefore, the capsule for-
mation, in most cases, can be possible only by adjusting the
volume fraction by trial and error.
To devise a building block preprogrammed to have a pre-
dictable self-assembly property for capsule formation, a
symmetrical CPFA with a C3-symmetric triskelion shape was
designed to allow the positioning of building blocks in a pre-
cise geometry for closed-capsule structure formation. CPFA
consists of even-numbered l-amino acids with alternating
hydrophilic and -phobic side chains (Figure 1).
In this type of a molecular arrangement, the side chains
(R groups at the a-carbon atoms) adopt alternating axial
and equatorial positions as a low-energy conformation. In
contrast, cyclic peptide structures made up of alternating l-
and d-amino acids, as shown in several synthetic cyclic pep-
tides,[11,25] can adopt a flat-ring structure as a low-energy
conformation in which all of the side chains lie horizontal to
the plane of the ring (equatorial). The six-residue CPFA
consists of three arginines and three hydrophobic amino
acids as hydrophilic and -phobic residues, respectively. The
hydrophilic and -phobic residues are placed at alternating
positions in order for the adjacent side chains to be located
at different faces (axial or equatorial).
Capsules or vesicles are one of the most important nano-
structural morphologies that can be utilized in many types
[a] E. K. Chung, Dr. E. Lee, Prof. Dr. M. Lee
Center for Supramolecular Nano-Assembly and
Department of Chemistry, Seoul National University
Seoul 151-747 (Korea)
The guanidinium group in arginine has been found to be
a crucial residue for cell-surface binding and penetration of
cell-penetrating peptides.[18,26,27] The hydrophobic amino
acids are tyrosine derivatives modified with long alkyl
chains. In addition to CPFA with the perhydrogenated alkyl
chain (hCPFA), CPFA with the perfluorinated alkyl chain
(fCPFA) was synthesized to take advantage of the “fluoro-
phobic effect”. The fluorophobic effect refers to the super-
Fax : (+82)2-393-6096
E-mail: myongslee@snu.ac.kr
[b] Prof. Dr. Y.-b. Lim
Department of Materials Science and Engineering
Yonsei University, Seoul 120-749 (Korea)
Fax : (+82)2-365-5882
E-mail: yblim@yonsei.ac.kr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200903145.
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The self-assembly behavior
of CPFA in aqueous solution
was investigated with dynamic
light scattering (DLS), circular
dichroism (CD) spectroscopy,
zeta-potential
measurements,
and transmission electron mi-
croscopy (TEM). DLS studies
revealed that hCPFA and
fCPFA self-assembled into
nanoaggregates with an average
hydrodynamic radii (RH) of 420
and 60 nm, respectively (Figur-
e 2aand Figure S1a in the Sup-
porting Information). CD and
FTIR results show that both
CPFAs are mostly in a random-
coil conformation, suggesting
that hydrogen-bonding interac-
tions responsible for peptide
secondary-structure formation
(e.g., a helix and b sheet) are
not involved in the self-assem-
bly process of CPFAs (Figur-
es S1b and S2 in the Supporting
Information). These results sug-
gest that the driving force for
the self-assembly of CPFAs is
pure hydrophobic interactions
between the perhydrogenated
or perfluorinated alkyl chains
rather than b-sheet interactions,
as found in nanotube-forming
cyclic peptides with alternating
l- and d-amino acids.[11,25] Anal-
ysis of nanostructural morphol-
ogy with TEM revealed that
hCPFA forms overall irregular
aggregates (Figure 2c), suggest-
ing
that
the
aggregation
strength of perhydrogenated
alkyl chains is rather weak. In
contrast, fCPFA self-assembled
into discrete nanocapsules, as
Figure 1. Structure and self-assembly of CPFA. a) Low-energy conformations of 6-residue cyclic peptides. Left: shown in Figure 2d. The con-
a cyclic peptide consists of only l-amino acids. Right: Molecular modeling of six-residue CPFAs (fCPFAs) by
Forcite geometry optimization. The Forcite calculation was performed by using the Accelrys Materials Studio
program. ax: axial, eq: equatorial. b) Self-assembly of fCPFAs into a capsule nanostructure.
caveness observed in the nega-
tively stained TEM image indi-
cates the shape of shrunken
capsules formed during the
drying process, providing the
hydrophobicity of fluorocarbon atoms and the selective self-
association between fluorinated moieties, which have been
demonstrated to be effective in stabilizing self-assembled
nanostructures and protein structures.[28–31] The head-to-tail
cyclization of the corresponding protected linear peptide is
conducted under pseudo-high-dilution conditions (for de-
tails, see the Supporting Information.)[32]
evidence of the hollow nature of the vesicular structures.
Moreover, the cryo-TEM image clearly shows the formation
of hollow nanocapsules with high contrast between the wall
and the hollow interior. Therefore, the strengthened hydro-
phobic interactions by the fluorophobic effect are important
for stabilizing the capsule nanostructures, which further sup-
port the notion that CPFAs self-assemble through purely hy-
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Cyclic Peptide Facial Amphiphile
COMMUNICATION
Figure 2. Self-assembly of CPFAs. a) DLS and b) CD analyses of fCPFA. Negatively stained TEM images of c) hCPFA and d) fCPFA. Inset in d): cryo-
TEM image. Samples (30 mm) were prepared in a 75 mm aqueous solution of KF.
drophobic interactions. The wall thickness (ca. 5.9 nm) cor-
responds well to the calculated thickness of the fCPFA bi-
layer (5.5 nm), indicating that the wall consists of the hydro-
phobic interior of alkyl chains and the hydrophilic exterior
of guanidinium groups (see Figure 1b). Zeta-potential (z)
measurement showed that fCPFA capsules had positive z
potential values ((+54Æ4) mV), which further demonstrates
the formation of the positively charged surface by the guani-
dinium group coating. All these data clearly show that C3-
symmetric and facially amphiphilic CPFAs predestined for
capsule formation do form the anticipated nanostructures.
To further demonstrate the importance of C3-symmetric
and cyclic properties of supramolecular building blocks for
round and closed-capsule nanostructure formation, analo-
gous molecules were designed and synthesized. The first an-
alogue (fLPA) is a linear amphiphilic peptide, which is a
precursor of fCPFA. As shown in Figure 3a, TEM analysis
shows the formation of nanosheets from fLPA in aqueous
solution. CD and FTIR experiments reveal the presence of
strong b-sheet hydrogen bonds in fLPA aggregates, indicat-
ing that the fLPA self-assembly process is not solely driven
by hydrophobic interactions (Figures S2 and S3 in the Sup-
porting Information). The result clearly illustrates that the
C3-symmetric and cyclic nature of the supramolecular build-
ing block is a necessary condition for capsule formation. To
further address the importance of a C3-symmetric structure
for capsule formation, fCPA was synthesized and its self-as-
sembly behavior was investigated (Figure 3b). fCPA has a
cyclic structure like that in fCPFA; however, fCPA has a C2-
symmetric structure, since the molecule has four arginines
and two amino acids with perfluorinated alkyl chains. Inves-
tigation of the nanostructural morphology with TEM reveals
that fCPA self-assembles into a bundle of nanofibers with
the diameter of the individual nanofiber being approximate-
Chem. Eur. J. 2010, 16, 5305 – 5309
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M. Lee, Y.-b. Lim et al.
Figure 3. Self-assembly of analogue building blocks. Negatively stained TEM images. Samples (30 mm) were prepared in a 75 mm aqueous solution of KF.
ly 5 nm. Again, this result demonstrates that, as in the case
of the clathrin protein and analogous triskelion molecules,
the building blocks should have C3-symmetry in order for
them to grow (self-assemble) in every direction.
and sonicated. The calcein-loaded nanocapsules were sepa-
rated from the free calcein by using gel filtration chromatog-
raphy with Sephadex G-25. Analyses of the nanocapsules by
DLS showed that the sizes of the nanocapsules after calcein
entrapment are almost similar to those of the original nano-
capsules (Figure 4a). TEM investigations revealed that the
spherical shape is preserved even after calcein entrapment
(inset in Figure 4a). The intracellular delivery experiment
was performed with a mammalian cell line, HeLa. The
result shows the successful intracellular delivery of calcein
(Figure 4b). The image shows the bright green fluorescence
of calcein in every cell, indicating the highly efficient intra-
cellular delivery capability of fCPFA nanocapsules.
In conclusion, novel and unique self-assembling peptide
building blocks, CPFAs, were designed, synthesized, and
their self-assembly behavior was investigated. The signifi-
cance of this approach lies in the fact that nanocapsule for-
mation could be predicted and controlled by the precise mo-
lecular design, rather than experimental adjustment of the
relative volume fraction as in amphiphilic block molecules.
The guanidinium-group-coated fCPFA developed in this
study should be useful for the intracellular delivery of cargo
molecules. Moreover, the results from this study indicate
that any type of hydrophilic-ligand-coated nanocapsules
The primary driving force underlying the formation of a
curved capsule structure is believed to be the combination
of the strong tendency of the C3-symmetric facial amphi-
phile to grow in a two-dimensional way and the lack of di-
rectional hydrogen-bonding interactions imposed by the
cyclic nature of the amphiphilic molecule. At the initial
stage of the self-assembly process, a 2D bilayered patch of a
certain size starts to bend to reduce its total energy, there-
after further addition of building blocks generates a closed-
capsule structure.[24,33] In that process, the absence of hydro-
À
gen-bonding interactions between amide C=O and N H
groups should play an important role, which would other-
wise complicate the self-assembly process.
Since an fCPFA nanocapsule has cell-binding/penetrating
guanidinium groups and a hollow interior, it might trap hy-
drophilic drugs and be developed as an intracellular nano-
carrier.[18,34] To explore such a possibility, entrapment experi-
ments were performed with a model hydrophilic compound,
calcein. For the entrapment of calcein within the nanocap-
sule, fCPFA and calcein were mixed in an aqueous solution
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Cyclic Peptide Facial Amphiphile
COMMUNICATION
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Acknowledgements
M.L. thanks a grant from the National Creative Research Initiative Pro-
gram of the National Research Foundation (NRF) of Korea. Y.-b.L.
thanks the NRF for support through Grants-in-aid 2009-0066736, 2009-
0076516, and 2009-0082617, and the grants from Translational Research
Center for Protein Function Control, Yonsei University (2009-0092971)
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Received: November 17, 2009
Revised: March 2, 2010
Published online: April 1, 2010
Keywords: amphiphiles · macrocycles · peptides · self-
assembly · vesicles
Chem. Eur. J. 2010, 16, 5305 – 5309
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5309