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: I.W.Hamley@reading.ac.uk

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

Dow

nloa

ded

by U

nive

rsity

of

Rea

ding

on

01 F

ebru

ary

2011

Publ

ishe

d on

29

Oct

ober

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/C

0CC

0379

3AView Online

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.

Dow

nloa

ded

by U

nive

rsity

of

Rea

ding

on

01 F

ebru

ary

2011

Publ

ishe

d on

29

Oct

ober

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/C

0CC

0379

3AView Online

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

1 H. G. Cui, M. J. Webber and S. I. Stupp, Pept. Sci., 2010, 94, 1.2 D. W. P. M. Lowik and J. C. M. van Hest, Chem. Soc. Rev., 2004,33, 234.

3 S. Cavalli and A. Kros, Adv. Mater., 2008, 20, 627.4 J. D. Tovar, R. C. Claussen and S. I. Stupp, J. Am. Chem. Soc.,2005, 127, 7337.

5 H. A. Behanna, J. Donners, A. C. Gordon and S. I. Stupp, J. Am.Chem. Soc., 2005, 127, 1193.

6 H. Cui, T. Muraoka, A. G. Cheetham and S. I. Stupp, Nano Lett.,2009, 9, 945.

7 E. T. Pashuck, H. Cui and S. I. Stupp, J. Am. Chem. Soc., 2010,132, 6041.

8 J. D. Hartgerink, E. Beniash and S. I. Stupp, Science, 2001, 294, 1684.9 E. D. Spoerke, S. G. Anthony and S. I. Stupp, Adv. Mater., 2009,21, 425.

10 G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. A. Harrington,J. A. Kessler and S. I. Stupp, Science, 2004, 303, 1352.

11 A. Mata, L. Hsu, R. Capito, C. Aparicio, K. Henrikson andS. I. Stupp, Soft Matter, 2009, 5, 1228.

12 M. A. Greenfield, J. R. Hoffman, M. O. de la Cruz and S. I. Stupp,Langmuir, 2010, 26, 3641.

13 R. N. Shah, N. A. Shah, M. M. D. Lim, C. Hsieh, G. Nuber andS. I. Stupp, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 3293.

14 M. O. Guler, L. Hsu, S. Soukasene, D. A. Harrington, J. F. Hulvatand S. I. Stupp, Biomacromolecules, 2006, 7, 1855.

15 Y. C. Yu, P. Berndt, M. Tirrell and G. B. Fields, J. Am. Chem.Soc., 1996, 118, 12515.

16 Y. C. Yu, M. Tirrell and G. B. Fields, J. Am. Chem. Soc., 1998,120, 9979.

17 D. W. P. M. Lowik, J. Garcia-Hartjes, J. T. Meijer and J. C. M.van Hest, Langmuir, 2005, 21, 524.

18 D. W. P. M. Lowik, I. O. Shklyarevskiy, L. Ruizendaal, P. C. M.Christianen, J. C. Maan and J. C. M. van Hest, Adv. Mater., 2007,19, 1191.

19 M. van den Heuvel, A. M. Prenen, J. C. Gielen, P. C. M.Christianen, D. J. Broer, D. Lowik and J. C. M. van Hest,J. Am. Chem. Soc., 2009, 131, 15014.

20 S. E. Paramonov, H.-W. Jun and J. D. Hartgerink, J. Am. Chem.Soc., 2006, 128, 7291.

21 S. Cavalli, J. W. Handgraaf, E. E. Tellers, D. C. Popescu,M. Overhand, K. Kjaer, V. Vaiser, N. Sommerdijk, H. Rapaportand A. Kros, J. Am. Chem. Soc., 2006, 128, 13959.

22 F. Versluis, H. R. Marsden and A. Kros, Chem. Soc. Rev., 2010,39, 3434.

23 H. H. Shen, R. K. Thomas, C. Y. Chen, R. C. Darton, S. C. Bakerand J. Penfold, Langmuir, 2009, 25, 4211.

24 D. J. Gordon, J. J. Balbach, R. Tycko and S. C. Meredith, Biophys.J., 2004, 86, 428.

25 B. Roper, D. Kaisig, F. Auer, E. Mergen and M. Molls,Strahlenther. Onkol., 2004, 180, 315.

26 Y. Y. Li, C. F. McTiernan and A. M. Feldman, Cardiovasc. Res.,2000, 46, 214.

27 F. X. Maquart, S. Pasco, L. Ramont, W. Hornebeck andJ. C. Monboisse, Crit. Rev. Oncol. Hematol., 2004, 49, 199.

28 K. T. Tran, L. Griffith and A. Wells,Wound Repair Regener., 2004,12, 262.

29 K. Katayama, J. Armendarizborunda, R. Raghow, A. H. Kangand J. M. Seyer, J. Biol. Chem., 1993, 268, 9941.

30 L. C. Serpell, Biochim. Biophys. Acta, 2000, 1502, 16.31 T. Miyazawa and E. R. Blout, J. Am. Chem. Soc., 1961, 83, 712.32 A. Barth and C. Zscherp, Q. Rev. Biophys., 2002, 35, 369.33 S. Krimm and J. Bandekar, Adv. Protein Chem., 1986, 38, 181.34 B. Stuart, Biological Applications of Infrared Spectroscopy, Wiley, 1997.35 C. R. Safinya, E. B. Sirota, R. F. Bruinsma, C. Jeppesen,

R. J. Plano and L. J. Wenzel, Science, 1993, 261, 588.36 C. R. Safinya, D. Roux, G. S. Smith, S. K. Sinha, P. Dimon,

N. A. Clark and A. M. Bellocq, Phys. Rev. Lett., 1986, 57, 2718.37 I. W. Hamley, V. Castelletto, O. O. Mykhaylyk, Z. Yang,

R. P. May, K. S. Lyakhova, G. J. A. Sevink andA. V. Zvelindovsky, Langmuir, 2004, 20, 10785.

Dow

nloa

ded

by U

nive

rsity

of

Rea

ding

on

01 F

ebru

ary

2011

Publ

ishe

d on

29

Oct

ober

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/C

0CC

0379

3AView Online