fibrillar superstructure from extended nanotapes formed by a ......this ournal is c the royal...
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This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 9185–9187 9185
Fibrillar superstructure from extended nanotapes formed
by a collagen-stimulating peptidew
Valeria Castelletto,a Ian W Hamley,z*a Javier Perez,b Ludmila Abezgauzc and
Dganit Daninoc
Received 10th September 2010, Accepted 8th October 2010
DOI: 10.1039/c0cc03793a
The nanostructure of a peptide amphiphile in commercial use in
anti-wrinkle creams is investigated. The peptide contains a
matrikine, collagen-stimulating, pentapeptide sequence. Self-
assembly into giant nanotapes is observed and the internal structure
was found to comprise bilayers parallel to the flat tape surfaces.
Peptide amphiphiles (PAs) comprise a lipid hydrophobic tail
connected to a hydrophilic peptide segment. This configuration
leads to self-assembled structures with a hydrophobic core
surrounded by a peptide functionalized corona. Self-assembly
commonly leads to nanofibres in which peptide epitopes are
presented on the fibril surface. These nanostructures are being
developed for diverse applications in bionanotechnology and
regenerative medicine.1–3 Stupp and coworkers have elucidated
the principles of self-assembly of PAs,4–7 and explored several
important applications in bioengineering, including the use of
PA nanofibers as scaffolds for biomineralization to regenerate
bone,8,9 in differentiation of progenitor10 or stem11 cells, or
tissue scaffolding12 including cartilage regeneration.13 PAs
enable the presentation of bioactive epitopes such as cell
adhesion motifs RGD8 or RGDS14 or IKVAV14 or cell growth
factors such as TGF-b1,13 at the surface of nanofibrils. In
general, the peptide amphiphiles have biologically relevant
lipid chain lengths, in particular palmitoyl (hexadecyl, C16).
Tirrell and coworkers probed the influence of alkyl chain
length on the self-assembly of PAs comprising C6 to C16 mono-
or di-alkyl chains linked to a collagen-mimetic peptide sequence,
forming a polyproline II-like triple helical structure.15,16 The
helical ordering was retained in the PA, and the thermal stability
of the collagen structure increased with alkyl chain length. van
Hest and coworkers have also investigated the influence of
(mono-alkyl) lipid chain length on the self-assembly of PAs
containing an octapeptide derived from a protein of the malaria
parasite P. falciparum. Short chains (C6–C12) produced random
coil structures, and b-sheet ordering was only observed for C14
and C16 derivatives, for which the C16 variant showed the
most extended thermal stability range for b-sheet structure.17
This group also demonstrated crosslinking and magnetic field
alignment of nanofibrils from polymerizable PAs comprising
diacetylene units in the lipid chain and the same peptide unit.18,19
Hartgerink and coworkers have systematically examined the
principles underpinning the self-assembly of peptide amphiphiles.20
The role of hydrogen bonding and amphiphilic packing on
self-assembly into nanofibrils has been explored, focusing on the
roles of specific residues in a series of designed variants.20–22 The
aggregation of the naturally occurring peptide amphiphile
surfactin (which has a cyclic peptide headgroup) at the air–water
interface has been investigated.23 The stability of monolayers at the
air–water interface showed that lipidation (using octanoyl chains)
enhances the amphiphilicity of peptide Ab(16–22) with sequence
KLVFFAE from the amyloid b (Ab) peptide.24
Here we investigate the self-assembly of the commercially
available PAmatrixyl, C16-KTTKS.25Matrixyls is the tradename
(registered to Sederma SA, Le Perray-en-Yvelines, France) for
this pentapeptide PA, part of the family of matrikines. These
are peptidic fragments involved in the natural process of
tissue repair.26–28 Peptide KTTKS promotes extracellular
matrix production and stimulates production of collagen and
fibronectin,29 helping to reform the skin’s extracellular matrix.
Lipidation presumably enhances bioavailability. Despite its
widespread use in anti-aging (anti-wrinkle) skin creams, we are
not aware of any prior published work on the self-assembly or
physico-chemical properties of C16-KTTKS. This is both of
intrinsic interest in view of previous research on PAs, and
should also shed light on its mode of action in cosmeceutical
applications. This is the first report to our knowledge on a
peptide amphiphile in current commercial use. A remarkable
self-assembled structure, hierarchically ordered from the
sub-nm to the mm scale has been uncovered for this PA.
Matrixyl forms a giant fibrillar superstructure at the tens of
microns length scale, as imaged by confocal microscopy and
differential interference microscopy (Fig. 1a and c). Branch points
(e.g., the one showing four thinner fibrils emerging from a thicker
one) are evident in Fig. 1a, indicating that they comprise smaller
subunits. In addition to giant tapes, Matrixyl also forms a smaller
population of cylindrical fibrils (Fig. S1 and S2, ESIw) as revealedby SEM and AFM. Fig. 1b shows that the fibrils formed by
Matrixyl take up the dye Congo red, a characteristic for amyloid
b-sheet fibril formation. The b-sheet secondary structure of the
peptide was also confirmed by FTIR, as discussed below.
The dense network of remarkable extended fibrils may act as
an excellent scaffold for collagen deposition, or in potential
applications in tissue growth.
The supramolecular self-assembled structure formed by
Matrixyl was further investigated at the nanoscale. Negative
aDept of Chemistry, University of Reading, Whiteknights, Reading,RG6 6AD, UK. E-mail: [email protected]
b Beamline SWING, Synchrotron Soleil, Orme-des-Merisiers,91190 Saint Aubin, France
cDepartment of Biotechnology and Food Engineering and the RussellBerrie Nanotechnology Institute, Technion-Israel Institute ofTechnology, Haifa 32000, Israelw Electronic supplementary information (ESI) available: Experimentalmethods, SEM, AFM, TEM images, CD spectra, XRD peak positions.See DOI: 10.1039/c0cc03793az Also at Diamond Light Source, Harwell Science and InnovationCampus, Chilton, Didcot, Oxfordshire OX11 0DE, UK.
COMMUNICATION www.rsc.org/chemcomm | ChemComm
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9186 Chem. Commun., 2010, 46, 9185–9187 This journal is c The Royal Society of Chemistry 2010
stain TEM and cryo-TEM images reveal extended tape
structures (Fig. 2a and b) with a persistence length extending
to tens of microns or more as shown in Fig. 1 and by AFM
and SEM (Fig. S1 and S2, ESIw) and considerable distribution
of tape width. The tape thickness estimated from AFM and
SEM images is 10–50 nm. The broad distribution of tape
widths is in contrast, for example, to previous reports on the
PA amphiphile C16-OVEVE.6 Cryo-TEM shows similar tape
structures for a 1 wt% solution (Fig. S3, ESIw).Interestingly, for lower concentration samples (0.075% in
water) twisting of the tapes was observed, as shown in Fig. S4
(ESIw). The twisted tapes have a lower polydispersity in width
than the flat tapes, ranging from 15�30 nm. This is probably
due to the lower width of the b-sheet laminates at the lower PA
concentration, thus enabling the tapes to twist. A large
energetic penalty to twisting is expected for very wide tapes.
Very occasionally, anisotropic crystallites were observed in
TEM images for heat-treated lower concentration samples
which seem to have a layered internal structure (Fig. S4, ESIw).X-Ray diffraction on an aligned stalk dried from a Matrixyl
solution shows a characteristic cross-b pattern with multiple
equatorial reflections and orthogonal meridional reflections
including a strong 4.78 A reflection corresponding to the
b-strand spacing (Fig. 2c). A full list of the d spacings associated
with the observed reflections can be found in Table S1 (ESIw). Thefirst observed equatorial reflection at 26.4 A corresponds closely to
the second order peak observed in the SAXS pattern for the
1 wt% solution (Fig. 3b). It was not possible to measure at lower
angle on the diffractometer used, prohibiting identification of the
probable first order peak for the dried stalk. Additional equatorial
reflections listed in Table S1 (ESIw) do not correspond to a simple
packing such as a one-dimensional or hexagonal lattice, and
indicate a more complex unit cell in the dried stalk. The main
4.78 A reflection is observed perpendicular to the equatorial
reflections, consistent with the spacing of b-strands in a cross-bX-ray diffraction pattern.30 The XRD pattern thus indicates
b-sheet ordering of the peptides. This was confirmed by FTIR
spectroscopy (Fig. 2d) which shows a strong peak in the amide I
region at 1609–1619 cm�1, the peakmoving to larger wavenumber
on increasing concentration. A subsidiary peak at around
1685 cm�1 associated with antiparallel b-sheet ordering31,32 is
absent, from which we deduce that the Matrixyl b-sheets are
parallel. The parallel ordering of the b-sheets is constrained by the
tethering of the peptide moieties to the lipid tails which are
arranged in a bilayer configuration (vide infra). The peak position
at 1609 cm�1 at low peptide concentration is slightly lower than
that typically observed for b-sheet structures and may reflect the
influence of the attached lipid chain and/or lysine side chain
features (NH3+ deformation) which can give bands at this
position.33,34 The presence of b-sheet structure is confirmed by
CD spectroscopy (Fig. S5), which shows the development of a
minimum at 217 nm which increases in depth with increasing
concentration (at low concentration, the CD spectra are
consistent with a significant content of disordered peptide).
Fig. 1 Fibrillar superstructure of peptide amphiphile Matrixyl. (a) Confocal optical microscopy image (fluorescent labelling with Rhodamine B,
0.014 wt% Matrixyl in water), (b) Apple green birefringence observed by polarized optical microscopy upon staining with Congo red,
(c) Differential optical microscopy image (1 mg mL�1 = 0.1 wt%).
Fig. 2 Extended nanotapes formed by self-assembly of Matrixyl,
(a) Negative stain TEM (sample dried from a 1 wt% solution), the top
narrow tape shows a striated internal structure, (b) Cryo-TEM (on a
0.1 wt% solution), the broad distribution of tape widths can be observed,
the tape persistence length exceeds microns (cf. Fig. 1), (c) XRD pattern
from a dried stalk showing a cross-b pattern consistent with b-sheetordering, (d) Amide I FTIR spectra showing b-sheet features.
Fig. 3 X-Ray data and model for Matrixyl self-assembly, (a) two-
dimensional SAXS data from a sample (0.95 wt%) aligned by
flow. The oriented Bragg reflections are nearly perpendicular to the
horizontal flow (v) direction and indicate alignment of PA bilayers
perpendicular to the flow direction (i.e. tapes align along the shear axis),
(b) one dimensional SAXS profiles showing Bragg peak positions, and
the development of diffuse scattering at high temperature, (c) structural
model for the PA bilayers.
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This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 9185–9187 9187
Small-angle X-ray scattering (SAXS) was used to probe the
nanoscale order of Matrixyl within the nanotapes. Fig. 3a
shows a SAXS pattern obtained from a 0.95 wt% sample after
injection into a capillary. The shear flow experienced by the
sample led to alignment. The SAXS pattern contains a series of
up to three (depending on temperature) equally spaced sharp
Bragg reflections, indicating a layer structure.35 The enhanced
orientation of the Bragg peaks perpendicular to the (horizontal)
flow direction indicate that the layer normal is perpendicular to
the flow direction, and to the X-ray beam, as shown in Fig. 3c.
Fig. 3b shows one-dimensional SAXS profiles obtained by
radial integration of two-dimensional SAXS patterns. The
location of the sharp Bragg peaks is evident, these correspond
to a layered structure with a period of 52.5 A. This indicates a
bilayer structure of the PA when the length of a hexadecyl alkyl
chain plus pentapeptide headgroup is considered. There are two
possible orientations of the bilayers consistent with the SAXS
data, depending on the orientation of the tapes with respect to
the shear plane. The most probable interpretation is that the
tapes comprise bilayers parallel to the faces of the tapes such
that the polar peptide moieties are presented at the tape surface.
In the other case, alternating lipid and peptide domains would
be presented at the tape surface. Cui et al. also proposed
a perpendicular arrangement of PAs with respect to the
tape surface, although without evidence from scattering
experiments.6 At high temperature (65 1C, expected to be above
the lipid chain melting temperature), some increase in diffuse
scattering is observed between the first and second Bragg peaks
and below the first Bragg peak. This is associated with an
increase in the number of defects within the layers,36,37 possibly
resulting from branching or breaking of the tapes above the
lipid chain melting transition (a discontinuity in intensity of CD
band intensities was also noted at around 65 1C). The internal
structure of the tapes with bilayers perpendicular to the faces
comprises hydrated channels containing the lipid headgroups.
This structure may be important in templating other bio-
molecules in applications. The molecular origin of the tape
superstructure is at present not clear in detail. The constrained
lateral dimensions (in particular, tape thickness) may originate
in the balance between the tendency of the lipid chains to form
an extended lamellar structure, and that of the peptides to form
(twisted) fibrillar structures based on b-sheets.6 There may also
be analogies with the end-cap energy of cylindrical micelles,
extended to the two-dimensional case of tapes, associated with
exposure of hydrophobic lipid chains to water. It is hoped that
our observations will stimulate theoretical work to examine this
in more detail.
In summary, this is the first report on the self-assembly of a
PA that has found an important application in anti-wrinkle
creams due to its activity in stimulating collagen production.
The macroscopic fibrillar structure is revealed, using multiple
techniques to characterize ordering on distinct length scales, to
comprise hierarchically ordered extended tapes with considerable
polydispersity in width (tens to hundreds of nanometres).
The tapes contain PA bilayers parallel to the faces. The
presentation of the peptide epitopes on the surface of the
tapes is presumably important in stimulating collagen produc-
tion. Understanding the self-assembled structure is important
in developing the next generation of collagen-stimulating
peptides for applications in skincare, wound healing and
regenerative medicine.
Notes and references
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