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Self-Assembly of PEGylated Peptide Conjugates Containing

a Modified Amyloid β-Peptide Fragment
V. Castelletto,* G. E. Newby Z. Zhu,† and I. W. Hamley‡
Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom

L. Noirez
CEA-CNRS Laboratoire L
eon Brillouin, F91191 Gif-sur-Yvette, France. †Currently at Department of
Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA. ‡Also at Diamond
Light Source, Chilton, Didcot, Oxfordshire OX11 0DE, United Kingdom.

Received January 9, 2010. Revised Manuscript Received April 26, 2010

The self-assembly of PEGylated peptides containing a modified sequence from the amyloid β peptide, FFKLVFF,
has been studied in aqueous solution. PEG molar masses PEG1k, PEG2k, and PEG10k were used in the conjugates. It is
shown that the three FFKLVFF-PEG hybrids form fibrils comprising a FFKLVFF core and a PEG corona. The β-sheet
secondary structure of the peptide is retained in the FFKLVFF fibril core. At sufficiently high concentrations,
FFKLVFF-PEG1k and FFKLVFF-PEG2k form a nematic phase, while PEG10k-FFKLVFF exhibits a hexagonal
columnar phase. Simultaneous small angle neutron scattering/shear flow experiments were performed to study the shear
flow alignment of the nematic and hexagonal liquid crystal phases. On drying, PEG crystallization occurs without
disruption of the FFKLVFF β-sheet structure leading to characteristic peaks in the X-ray diffraction pattern and FTIR
spectra. The stability of β-sheet structures was also studied in blends of FFKLVFF-PEG conjugates with poly(acrylic
acid) (PAA). While PEG crystallization is only observed up to 25% PAA content in the blends, the FFKLVFF β-sheet
structure is retained up to 75% PAA.

Introduction conjugate and can enhance in vivo circulation or residence

Peptide/polymer conjugates are the focus of immense interest
because it is possible to combine synergistically the properties of
peptides (functionality, responsiveness) with those of synthetic
polymers (solubility, responsiveness, cheapness).1-8 Short pep-
tides attached to polymers have been shown to be capable of
guiding the polymer superstructure.6-8 This could become a
powerful method to control polymer morphology and properties,
using biologically inspired end/side groups. From the other
perspective, polymers may be used as supports to present peptide
functionality within polymeric matrices (gels) or to guide the
ordering of peptide units via amphiphilic-type self-assembly.
Several recent reviews focus on polymer/peptide conjugates and
discuss different approaches to their synthesis using convergent
and divergent methods, based on growth of polymer from tethered
peptides, or vice versa, or coupling of presynthesized units.3,5,9-12
Conjugation of PEG to peptides and proteins is of great
interest because PEG provides a neutral, water-soluble polymeric
coating around the biomolecule that can reduce uptake of the
(1) L€owik, D. W. P. M.; Ayres, L.; Smeenk, J. M.; van Hest, J. C. M. Adv.
Polym. Sci. 2006, 202, 19–52.
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(5) Gauthier, M. A.; Klok, H.-A. Chem. Commun. 2008, 2591–2611.
(6) B€orner, H. G. Prog. Polym. Sci. 2009, 34, 811–851.
(7) Klok, H.-A. J. Polym. Sci.: Polym. Chem. 2005, 43, 1–17.
(8) B€orner, H. G.; Kuehnle, H.; Hentschel, J. J. Polym. Sci.: Polym. Chem. 2010,
48, 1–14.
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(12) Lutz, J. F.; B€orner, H. G. Prog. Polym. Sci. 2008, 33, 1–39.
time.1,7,12-17 A nice review discusses applications of PEGylation
of peptides and proteins for applications in biotechnology.18
From a more fundamental viewpoint, it is of interest to examine
whether the secondary structure of the polypeptide is influenced
when linked to PEG. From another perspective, these conjugates
may exhibit amphiphilic behavior similar to that of conventional
surfactants. Nonionic surfactants often contain poly(ethylene glycol)
(PEG) conjugated to a short hydrophobic moiety (commonly an
alkyl chain with six to eighteen carbon atoms).
The influence of polymer chain length on the self-assembly of
polymer/peptide conjugates has previously been investigated by
several groups. Klok and co-workers have investigated the self-
assembly of PEG-peptides containing bioinspired coiled coil
peptide sequences.19,20 The conjugates prepared had PEG molec-
ular weights of either 750 g mol-1 or 2000 g mol-1. The focus was
on the stabilization of the coiled coil peptide structure against pH,
concentration, and temperature changes conferred by PEG. In
general, little effect of PEG molar mass was noted when pH was
varied. In the case of varying peptide concentration, the higher
(13) Delgado, C.; Francis, G. E.; Fisher, D. Crit. Rev. Ther. Drug 1992, 9, 249–
304.
(14) Zalipsky, S. Adv. Drug Deliver. Rev. 1995, 16, 157–182.
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551.
(16) Veronese, F. M. Biomaterials 2001, 22, 405–417.
(17) B€orner, H. G.; Smarsly, B.; Hentschel, J.; Rank, A.; Schubert, R.; Geng, Y.;
Discher, D. E.; Hellweg, T.; Brandt, A. Macromolecules 2008, 41, 1430–1437.
(18) Ryan, S. M.; Mantovani, G.; Wang, X.; Haddleton, D. M.; Brayden, D. J.
Expert Opin. Drug Delivery 2008, 5, 371–383.
(19) Vandermeulen, G. W. M.; Tziatzios, C.; Duncan, R.; Klok, H.-A. Macro-
molecules 2005, 38, 761–769.
(20) Klok, H.-A.; Vandermeulen, G. W. M.; Nuhn, H.; Rosler, A.; Hamley,
I. W.; Castelletto, V.; Xu, H.; Sheiko, S. S. Faraday Discuss. 2005, 128, 29–41.

9986 DOI: 10.1021/la100110f Published on Web 05/07/2010 Langmuir 2010, 26(12), 9986–9996
Castelletto et al.
molar mass PEG led to a lower helix content20 or to enhanced
stability against pH change in the case of a heteropeptide
conjugate containing oppositely charged residues designed to
favor electrostatic interpolyelectrolyte complex formation.19
B€orner and co-workers have investigated polymer-peptide con-
jugates based on poly(n-butyl acrylate) (PnBA) of varying mo-
lecular weight conjugated to a β-sheet peptide (TV)5 aggregating
domain peptide.4 The dimensions of fibrillar aggregates imaged
by AFM were found to increase with PnBA chain length, and the
kinetics of self-assembly in solution were found to be retarded for
the highest molar mass sample (PnBA 38k). Lynn and co-workers
have investigated the self-assembly of conjugates of PEG with an
amyloid β (Aβ) peptide fragment, Aβ(10-35)-PEG3k,21 but did
not examine the influence of PEG molar mass. Conjugation to
PEG was found to enhance the solubility of the Aβ peptide, and
led to reversible fibrillization, in contrast to the native peptide.
We have recently investigated the self-assembly of PEG/pep-
tides containing peptides based on the sequence KLVFF, Aβ-
(17-20), from the amyloid beta (Aβ) peptide. This core sequence
has been shown to be important in fibrillization.22 We have
investigated the self-assembly in aqueous solution of FFKLVFF-
PEG3k (PEG3k denotes PEG with approximate Mn = 3000 g mol-1)
and found that it forms lyotropic liquid crystal phases at
sufficiently high concentration in water.23-25 We have also
shown that PEG3k crystallizes when FFKLVFF/PEG3k con-
jugates are dried at room temperature, leading to an interplay
between PEG crystallization and peptide fibrillization.26,27 In
the present paper, the influence of PEG chain length and
position is investigated, using again the model FFKLVFF
sequence used in our previous work. The peptide FFKLVFF
is itself hydrophobic, indeed does not dissolve in water; however,
when conjugated to PEG it provides a suitable hydrophile/
lipophile balance such that FFKLVFF-PEG conjugates show
excellent amphiphilic properties. Here, we study conjugates with
PEG1k or PEG2k attached at the C terminus as in our previous
study of PEG3k, and also PEG10k, which for synthetic reasons
was attached at the N terminus of the peptide. We show that
these samples also exhibit lyotropic liquid crystallinity; in
particular, nematic and hexagonal columnar phases are ob-
served depending on concentration and PEG chain length. We
also investigate the influence of PEG molar mass on the
morphology in dried samples;only PEG of sufficient molecular
weight is capable of crystallizing. Finally, the influence of
addition of an associating polymer, poly(acrylic acid) (PAA),
which can form a hydrogen-bonded complex with PEG,28 was
examined. This was motivated by an attempt to increase the
degree of segregation between the PEG and peptide domains,
with the objective of producing microphase separation in the
melt state. This was not achieved under the conditions studied;
nonetheless, the effect of PAA on the structure in the solid state
was probed. Specifically, the influence of PAA on PEG crystal-
lization and cross-β fibril structure was examined as a function
of blend composition.
(21) Burkoth, T. S.; Benzinger, T. L. S.; Jones, D. N. M.; Hallenga, K.;
Meredith, S. C.; Lynn, D. G. J. Am. Chem. Soc. 1998, 120, 7655–7656.
(22) Hamley, I. W. Angew. Chem., Int. Ed. Engl. 2007, 46, 8128–8147.
(23) Hamley, I. W.; Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Noirez, L.;
Hule, R. A.; Pochan, D. Chem.;Eur. J. 2008, 14, 11369–11374.
(24) Hamley, I. W.; Krysmann, M. J.; Castelletto, V.; Noirez, L. Adv. Mater.
2008, 20, 4394–4397.
(25) Hamley, I. W.; Krysmann, M. J.; Newby, G. E.; Castelletto, V.; Noirez, L.
Phys. Rev. E 2008, 77, 062901.
(26) Hamley, I. W.; Krysmann, M. J. Langmuir 2008, 24, 8210–8214.
(27) Krysmann, M. J.; Hamley, I. W.; Funari, S. S.; Canetta, E. Macromol.
Chem. Phys. 2008, 209, 883–889.
(28) Tirumala, V. R.; Ilavsky, J.; Ilavsky, M. J. Chem. Phys. 2006, 124, 234911.
Langmuir 2010, 26(12), 9986–9996
Article
Experimental Section
Materials. The FFKLVFF/PEG conjugates FFKLVFF-
PEG1k and FFKLVFF-PEG2k were synthesized by Rapp poly-
mere GmbH (T€ ubingen, Germany) using solid-phase peptide
synthesis methods, and were supplied as HCl salts. The synthesis
method is similar to that used to prepare YYKLVFF-PEG
polymers already reported by us.29
The conjugates FFKLVFF-PEG1K and FFKLVFF-PEG2K
were characterized (by the supplier) by reverse-phase high-per-
formance liquid chromatography (RP-HPLC; Grom Saphir 200,
C18 5 μm column). A mobile phase of a gradient of water with
0.1% TFA and acetonitrile with 0.75% TFA was used to confirm
high purity. Sample elution was monitored using a UV/vis
detector operating at 220 nm.
MALDI-TOF (Ultraflex, Bruker with matrix Universalmatrix,
Fluka) as performed by the supplier was used to confirm Mw =
1895 g mol-1 for FFKLVFF-PEG1k, while GPC provided Mn =
1046.8 g mol-1 for the precursor PEG. MALDI-TOF confirmed
Mw = 3095 g mol-1 (Mw/Mn < 1.05) for FFKLVFF-PEG2k and
Mn = 2103 g mol-1 for the precursor PEG.
PEG10k-FFKLVFF was obtained from American Peptide
Inc. (Sunnyvale, USA) as a TFA salt. The peptide fragment (Mpr-
Phe-Phe-Lys-Leu-Val-Phe-Phe) was synthesized on Fmoc-Phe-
Wang resin by using standard Fmoc/tBu chemistry. Protecting
groups used for amino acids are as follows: Trt group for Mpr and
Boc for Lys. Fmoc-protected amino acids were purchased from
EMD Biosciences and GL Biochem. Reagents for coupling and
cleavage were purchased from Aldrich. Solvents were purchased
from Fisher Scientific. The peptide chain was assembled on resin
by repetitive removal of the Fmoc protecting group by treating
with 20% piperidine/DMF for 30 min and coupling of protected
amino acid with HBTU/HOBt/NMM for 90 min. Ninhydrin
testing was performed after each coupling to check the coupling
efficiency. After the last coupling, the resulting resin was washed
and dried, then treated with reagent K (TFA/thioanisole/phenol/
EDT/water, 87.5:5:2.5:2.5:2.5, v/v) 3 h for cleavage and removal
of the side chain protecting groups. Crude peptide was precipi-
tated from cold ether and collected by filtration. The peptide
fragment was purified by reverse-phase HPLC to 95%, pooled
fractions were lyophilized to dry. Yield was 108 mg. MS of the
peptide before PEG10k conjugation provided Mw = 1035.3 g mol-1.
The above peptide substrate (108 mg, 0.1 mmol) was then
conjugated with 1 equiv PEG10K-Maleimide (1000 mg, 0.1 mmol)
in aqueous solution at pH 8. GPC provided Mn = 9531 g mol-1
and Mw = 9872 g mol-1 (PDI 1.04) for the precursor PEG10k.
The PEGylated product was further purified by reversed-phase
HPLC to 95%, and the pooled fractions were lyophilized to dry.
Poly(acrylic acid) PAA20 (Mw = 21 800 g mol-1, Mn = 20 000 g
mol-1) and PAA88 (Mw = 98 500 g mol-1, Mn = 88 000 g mol-1)
were purchased from Polymer Source Inc. (Quebec, Canada) and
used as received.
Peptide solutions were prepared by mixing weighed amounts of
peptide and Milli-Q water and allowing the sample to mix by
diffusion over a period of ∼7 days. Peptide/polymer blends were
made by codissolving them at a given weight ratio in water. After
spontaneous solvent evaporation, the blends were annealed at
80 °C for 24 h. Allowing for solvent evaporation and the
subsequent annealing at 80 °C provided thin films of blends, used
for XRD studies.
Fourier Transform Infrared (FTIR) Spectroscopy. Spec-
tra were measured on a Nicolet Nexus spectrometer with DTGS
detector. Solutions of FFKLVFF-PEG1k, FFKLVFF-PEG2k,
and PEG10k-FFKLVFF in D2O (0.9, 2, 3.2, 3.6, 5, 6.9, 7.7, 11.3,
and 20 wt %) were sandwiched between two CaF2 plate windows
(spacer 0.006 mm). Spectra were scanned 128 times over the range
4000-900 cm-1.
(29) Castelletto, V.; Newby, G. E.; Hermida-Merino, D.; Hamley, I. W.; Liu, D.;
Noirez, L. Polym. Chem. 2009, in press.
DOI: 10.1021/la100110f 9987
Article
Raman Spectroscopy. Raman spectra were recorded using a
Renishaw inVia Raman microscope. The light source was a
multiline laser, such that the experiments were performed using
the λ = 785 nm edge. Experiments were made on stalks prepared
by drying filaments of the peptide obtained from a 10 wt %
PEG10k-FFKLVFF sample. The stalks were focused by using a
20
magnification lens. Spectra were obtained in the interval
(100-3000) cm-1, using 20 s collection time with 10% laser power
and taking two averages.
Circular Dichroism (CD). Spectra were recorded using a
Chirascan spectropolarimeter (Applied Photophysics, UK). CD
was performed using FFKLVFF-PEG1k, FFKLVFF-PEG2k,
or PEG10k-FFKLVFF dissolved in water (0.05, 0.06, 0.9, and
1 wt %) and loaded into quartz coverslip cuvettes (0.1-mm-thick)
or into 1-mm-thick quartz bottles. Spectra are presented with
absorbance A < 2 at any measured point with a 0.5 nm step, 1 nm
bandwidth, and 1 s collection time per step at 20 °C.
Fluorescence Spectroscopy. Spectra were recorded using a
Cary Eclipse Varian Fluorescence Spectrometer with samples in a
1.0 cm quartz cuvette. Spectra were measured for samples in
water (0.005 or 0.007 wt %). The spectra were recorded from 279
to 490 nm using an excitation wavelength λex = 265 nm.
Cryogenic-Transmission Electron Microscopy (Cryo-TEM).
Experiments were performed at Unilever Research, Colworth
(Bedford, UK). Solutions of FFKLVFF-PEG1k, FFKLVFF-
PEG2k, or PEG10k-FFKLVFF (1.5, 1.8, 1.9 wt %) were blotted
and vitrified using a Gatan Cp3 cryoplunge system. Samples were
prepared at a controlled temperature of 22 °C and at a relative
humidity around 90%. A 3 μL drop of the solution was placed on
a 400-mesh copper TEM grid (Agar) covered with a per-
forated carbon film (plasma-treated). The drop was automatically
blotted, and the sample was plunged into liquid ethane (-183 °C)
to form a vitrified specimen,30,31 then transferred to liquid nitro-
gen (-196 °C) for storage. Specimens were examined in a JEOL
JEM-2100 electron microscope at 200 kV, at temperatures below
-175 °C. Images were recorded digitally on a Gatan UltraScan
1000 cooled CCD camera using DigitalMicrograph (Gatan) in the
low-dose imaging mode to minimize beam exposure and electron-
beam radiation damage.
Small-Angle Neutron Scattering (SANS). Small-angle
neutron scattering (SANS and rheo-SANS) was performed on
6.9 wt % FFKLVFF-PEG1k, 7.7 wt % FFLVFF-PEG2k, and
10.9 wt % PEG10k-FFKLVFF samples, using the 2D sensitive
multidetector PAXY of the Laboratoire L
eon Brillouin. Samples
were placed in a quartz Couette cell (0.1 mm gap) which was used
to apply steady shear.32 Shear rates are defined as γ_ = ΩRh/(R0 -
R1), where Ω is the angular velocity, Rh is the average radius, and
R0 = 19.0 mm and R1 = 19.1 mm are the inner and outer radii.
Shear rates applied were γ_ = 0.1-5 s-1. Measurements were per-
formed at room temperature. Data were obtained with neutrons
incident along the shear gradient direction (radial configuration).
A wavelength of 6 Å was used. The sample-detector distance
was fixed at 2.5 m. The corresponding q range extends from 0.023
to 0.129 Å-1.
Polarized Optical Microscopy (POM). Microscopy experi-
ments were performed by placing the sample between crossed
polarizers in an Olympus BX41 polarized microscope. FKLVFF-
PEG1k (6.9 wt %), FFKLVFF-PEG2K (7.8 wt %), and PEG10k-
FFKLVFF (20 wt %) samples were placed between a glass slide
and a coverslip before capturing the images with a Canon G2
digital camera. A few drops of 11.5 wt % PEG10k-FFKLVFF
were also placed on a glass slide and left to dry before capturing the
image with a Canon G2 digital camera.
(30) Talmon, Y. (1999) Cryogenic transmission electron microscopy in the study of
surfactant systems, In Modern Characterization Methods of Surfactant Systems
(Binks, B. P., Ed.), pp 147-178, Marcel Dekker, New York.
(31) Cui, H.; Hodgdon, T. K.; Kaler, E. W.; Abezgaous, L.; Danino, D.;
Lubovsky, M.; Talmon, Y.; Pochan, D. J. Soft Matter 2007, 3, 945–955.
(32) Baroni, P.; Pujolle, C.; Noirez, L. Rev. Sci. Instrum. 2001, 72, 2686.
9988 DOI: 10.1021/la100110f
Castelletto et al.
X-ray Diffraction (XRD). The experiments were performed
using a RAXIS IVþþ X-ray diffractometer (Rigaku) equipped
with a rotating anode generator. The XRD data was collected
using a Saturn 992 CCD camera. Diffraction patterns for the
pure peptides were obtained for stalks prepared by drying fila-
ments of the peptide. Aqueous solutions of FFKLVFF-PEG1k
(6.9 wt %), FFKLVFF-PEG2k (7.7 wt %), and PEG10k-
FFKLVFF (10.9 wt %) were suspended between the ends of a
wax-coated capillary and dried. Thin films of blends contain-
ing the FFKLVFF/PEG conjugate and (0-100) % PAA20k or
(0-100) % PAA88k were also studied by XRD. The stalks or the
thin films were mounted (vertically) onto the four axis goniometer
of the X-ray diffractometer.
Rheology. Rheological properties were determined using a
controlled-stress TA Instruments AR-2000 rheometer (TA In-
struments). For a fluid 5 wt % solution of FFKLVFF-PEG2k, a
Mooney geometry was used. For a gel-like 10 wt % sample, a
cone-and-plate geometry (cone diameter 20 mm, angle 1°) was
used. Frequency sweeps were performed at 25 °C. Preliminary
strain sweeps were performed for each sample in order to define
the linear viscoelastic region, thus ensuring that moduli were
independent of strain.
Differential Scanning Calorimetry (DSC). Melting points
for the FFKLVFF/PEG conjugates and the glass transition
temperature of the PAA were measured by DSC using a Mettler
DSC 823 system, at heating rates of 2 °C/min and 10 °C/min,
respectively.
Results
Self-Assembly in Solution and Secondary Structure of
FFKLVFF/PEG Conjugates. An important question is whether
the secondary structure of the peptide is retained in the PEG/
peptide conjugate. This was investigated by FTIR and CD
spectroscopy.
The secondary structure of the self-assembled peptides in
solution was first studied by FT-IR in the transmission config-
uration. Figure 1a-c show the Amide I and Amide II regions of
the FTIR spectra measured for D2O solutions of the three
FFKLVFF/PEG conjugates.
The FTIR spectra for (0.9-6.9) wt % FFKLVFF-PEG1k
(Figure1a) exhibit maxima at 1682 and 1618 cm-1. The max-
imum at 1618 cm-1 becomes more intense upon increasing
concentration, compared to the peak at 1682 cm-1. Figure 1b
shows that, while the FTIR spectra for 0.9 wt % FFKLVFF-
PEG2k shows only one well-defined peak at 1617 cm-1, the
FTIR spectra for 3.6 and 7.7 FFKLVFF-PEG2k present three
maxima at 1682, 1672, and 1617 cm -1.
Figure 1c shows that the FTIR spectra for 2-20 wt % PEG10k-
FFKLVFF present two maxima at 1700 and 1674 cm-1 and a third
maximum which shifts from 1625 to 1631 cm-1 upon increasing
concentration The intensity of the peak at (1625-1631) cm-1
increases upon increasing the concentration, while the remaining
two peaks are nearly insensitive to the concentration. It also is
noticeable that a peak at 1553 cm-1 starts to develop for 11.3 wt %
peptide and becomes clear in the spectrum for 20 wt % peptide.
The FTIR spectra in Figure 1a-c provide comparative in-
formation about the secondary structure of the FFKLVFF/PEG
conjugates, as a function of the PEG length and the sample
concentration.
FTIR peaks at (1617-1631) cm-1 are associated with a β-sheet
structure.33,34 The simultaneous presence of peaks at (1617-1631)
cm-1 and at (1682 -1700) cm-1 suggests an antiparallel β-sheet
(33) Haris, P.; Chapman, D. Biopolymers 1995, 37, 251–263.
(34) Stuart, B. (1997) Biological Applications of Infrared Spectroscopy; Wiley:
Chichester.
Langmuir 2010, 26(12), 9986–9996
Castelletto et al.
Figure 1. Amide I/II regions of FTIR spectra for samples in D2O at the concentrations indicated: (a) FFKLVFF-PEG1k, (b) FFKLVFF-
PEG2k, and (c) PEG10k-FFKLVFF. (d) FTIR for 11.3 wt % PEG10k-FFKLVFF showing peaks corresponding to semicrystalline PEG.
arrangement (Figure 1).33-36 The observed FTIR spectra also
contain contributions from TFA, corresponding to the peak at
(1672-1674) cm-1 (Figure 1b,c). The TFA content in FFKLVFF-
PEG2k solutions might consist of residual solvent from the HPLC
process, while the PEG10k-FFKLVFF was provided as a TFA salt
by the manufacturer. The peak at 1533 cm-1 measured in Figure 1c
is associated with the amide II band,34,37 and arises from the
N-H in-plane bending or C-N stretching modes of the amide
backbone.38
According to our previous studies,23,24 FFKLVFF-PEG3k
self-assembles into fibrils in aqueous solution at concentrations
similar to those studied in Figure 1. The fibrils consist of a
hydrophobic core containing the FFKLVFF block, surrounded
by a hydrophilic corona containing the PEG block. The FFKLVFF
block is arranged in β-sheet strands within the fibril hydrophobic
core.
In good agreement with previous data for FFKLVFF-PEG3k
fibrils,23,24 the FTIR results in Figure 1 show the existence of
antiparallel β-sheet structure in solutions of FFKLVFF-PEG1k,
FFKLVFF-PEG2k, and PEG10k-FFKLVFF, such that the
population of β-sheets increases upon increasing the concentra-
tion of the sample, at least for the former two samples. The spectra
for PEG10k-FFKLVFF are more complex, which may reflect an
increased influence of the PEG chain on the ordering of the
peptide as discussed below in the context of results from CD
spectroscopy.
Features in the FTIR spectra in the region 1370-1000 cm-1
can provide information about PEG crystallization. Indeed, only
the FTIR spectra for the FFKLVFF/PEG conjugates containing
PEG2k and PEG10k show some features corresponding to the
ordering of the PEG block in solution. A representative example
(35) Rosler, A.; Klok, H.-A.; Hamley, I. W.; Castelletto, V.; Mykhaylyk, O. O.
Biomacromolecules 2003, 4, 859–863.
(36) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712–719.
(37) Lin, S.-Y.; Chu, H.-L. Int. J. Biol. Macromol. 2003, 32, 173–177.
(38) Sarkar, S.; Chourasia, A.; Maji, S.; Sadhukran, S.; Kumar, S.; Adhikari, B.
Mater. Sci. 2006, 29, 475–484.
Langmuir 2010, 26(12), 9986–9996
Article
is shown in Figure 1d for 11.3 wt % PEG10k-FFKLVFF. The
spectrum contains peaks at 1035, 1090, 1139, 1254, 1332, and
1350 cm-1 which are usually associated with semicrystalline
PEG.39 These results indicate that the length of the PEG block
in FFKLVFF-PEG2k and PEG10k-FFKLVFF allows for its
ordering in solution, but does not disrupt the stability of the
β-sheet structure.
The crystallization of the PEG block will be addressed later
in this work regarding samples dried from solutions of the
FFKLVFF/PEG conjugates.
CD was used to probe changes in the secondary structure of the
peptide self-assembly, as a function of concentration and the PEG
block length. Figure 2a shows the CD spectra obtained for
solutions containing (0.05-0.06) wt % FFKLVFF/PEG conju-
gates, while Figure 2b contains CD results for samples containing
1 wt % FFKLVFF/PEG conjugates.
The CD spectra for the dilute solutions in Figure 2a are domi-
nated by a maximum at ∼218 nm and a minimum at ∼232 nm.
A prominent single maximum at ∼220 nm and a minimum at
230 nm have been previously reported by us in the CD spectrum
for FFKLVFF-PEG3k,23 FFKLVFF,40 and FFFF-PEG3k.41
According to our previous work, and in agreement with CD
results in the literature for phenylalanine oligopeptides,42 a strong
positive peak at 218 nm may result from the π-π* stacking. On
the other hand, spectra resembling those in Figure 2 reported for
peptide amphiphiles lacking aromatic residues have also been
ascribed to a coexistence of β-sheet ordering of peptide at the core
of the fibril with polyproline II type ordering of strands at the
periphery.43 A complex variation in the conformation of the
(39) Zheng, Y.; Bruening, M. L.; Baker, G. L. Macromolecules 2007, 40, 8212–
8219.
(40) Krysmann, M. J.; Castelletto, V.; Hamley, I. W. Soft Matter 2007, 2, 1401–
1406.
(41) Castelletto, V.; Hamley, I. W. Biophys. Chem. 2009, 141, 169–174.
(42) Peggion, E.; Palumbo, M.; Bonora, G. M.; Toniolo, C. Bioorg. Chem. 1974,
3, 125.
(43) Paramonov, S. E.; Jun, H. W.; Hartgerink, J. D. J. Am. Chem. Soc. 2006,
128, 7291–7298.
DOI: 10.1021/la100110f 9989
Article
Figure 2. Molar ellipticity for solutions of (a) (0.05-0.06) wt %
and (b) (0.9-1) wt % FFKLVFF/PEG conjugates.
strand as a function of distance from the junction point may
likewise be anticipated in our PEG/peptide conjugates.
The inset in Figure 2a shows the ellipticity values at ∼218 nm
(region A) and at ∼232 nm (region B) as a function of the PEG
length. The ellipticity at 232 nm is weakly sensitive to the PEG
length, while the ellipticity at 218 increases with PEG length. It is
possible that this indicates a decrease in β-sheet ordering upon
increasing the PEG chain length. This is not unreasonable
considering that a lengthy PEG chain may be expected to more
strongly influence the packing, in this case disrupting the β-sheet
hydrogen bonding, of more of the residues in the peptide.
Figure 2b shows that for 1 wt % FFKLVFF/PEG conjugates
there is an evolution in the CD spectra as a function of the PEG
block length. The CD spectrum for 0.9 wt % FFKLVFF-PEG1k
has negative bands centered at 215 and 222 nm. The minimum at
215 nm is associated with a β-sheet structure. The β-sheet
minimum becomes gradually canceled by a positive band which
starts to grow at 219 nm in the CD spectra, upon increasing the
PEG length from PEG1k to PEG10k (Figure 2b). The minimum
at 222 nm observed for PEG1k gradually shifts to 226 and 230 nm
for PEG2k and PEG10k, respectively.
The CD signal for samples containing PEG1k in Figure 2b is
dominated by the β-sheet structure of the fibrils, in good agree-
ment with FTIR results in Figure 1a. For longer PEG chains, the
growth of a positive band at 219 nm reflects the increased
influence of PEG on residues close to the junction point for
which the β-sheet ordering is disrupted, possibly replaced by
polyproline II ordering, as discussed by Paramonov for peptide
amphiphiles (in which the core comprises hydrophobic lipid and
the shell is peptide, i.e., the inverse structure to our system).43
These observations may shed light on the complex FTIR spectra
shown in Figure 1, although analysis of these in terms of the
variable ordering of residues as a function of distance from the
PEG junction point is complex.
The inset in Figure 2b shows the ellipticity values in regions (A)
and (B) of the CD spectra increase with PEG length. Phenylalanine
9990 DOI: 10.1021/la100110f
Castelletto et al.
associations become the fingerprint of the CD pattern upon
increasing the PEG length, while the minimum of the β-sheet is
shifted to higher wavelengths enhancing the value of the mini-
mum corresponding to the n-π* transition in the CD spectra.
Fluorescence spectroscopy confirmed the role of aromatic
stacking interactions between phenylalanine residues in the self-
assembly process. Figure S1 (in the Supporting Information)
presents spectra for dilute aqueous solutions of the three
FFKLVFF/PEG conjugates. The emission peak at ∼305 nm
is associated with π-π* stacking interactions between the
phenylalanine residues, as discussed elsewhere.44
Evidence for self-assembly into fibrils is provided by cryo-
TEM. Cryo-TEM was performed instead of conventional nega-
tive stain TEM, due to the fact that PEG can crystallize when
drying the sample. Cryo-TEM enables the in situ structure to be
trapped in vitrified water, circumventing the crystallization of
PEG.26 Figure 3 shows images obtained for all three conjugates
for 1.5-1.9 wt % solutions. Figure 3 shows that FFKLVFF/PEG
conjugates form fibrils with a well-defined diameter d = (5.7 (
0.7) nm, (5.2 ( 0.8) nm, and (5.6 ( 0.6) nm for FFKLVFF-
PEG1k, FFKLVFF-PEG2k, and PEG10k-FFKLVFF, respec-
tively. Since the thickness of the FFKLVFF/PEG conjugate
fibrils in Figure 3 does not depend on the PEG length, it is likely
that the structure imaged by the cryo-TEM corresponds only to
the hydrophobic FFKLVFF core of the fibrils. Furthermore, no
contrast between core and shell was observed, indicating that the
core (and possibly adjacent high density inner shell region) only
are imaged.
The length of the fibers in Figure 3 is rather polydisperse,
extending up to several micrometers (based on these images,
and several others obtained for these samples). It has to be
pointed out that, for a similar concentration of FFKLVFF/
PEG conjugate, the number density of long fibres in the cryo-
TEM images decreases upon increasing the PEG length. It is
possible that excluded volume interactions between PEG cor-
onas of neighboring fibril, increase the distance between fibrils
upon increasing the PEG length, hence reducing the number
density of fibrils.
In summary, FTIR and cryo-TEM results indicate that
the FFKLVFF/PEG conjugates self-assemble into fibrils with a
β-sheet structure at concentrations as low as 1 wt % FFKLVFF/
PEG conjugate. CD and fluorescence results suggest the forma-
tion of aggregates in solution for (0.005-0.06) wt % FFKLVFF/
PEG conjugate. Although our results do not provide evidence
about the geometry of the self-assembled structure of the aggre-
gates at low concentration, it is certain that interactions between
neighboring phenylalanine units play an important role in their
structure.
Liquid Crystal Phase Formation and Dynamics of Shear
Flow Alignment for FFKLVFF/PEG Conjugates. Liquid
crystal phase formation was noted at higher concentration, via
characteristic birefringence textures observed by polarized optical
microscopy. Figure S2 (Supporting Information) shows the
birefringence textures obtained for 6.9 wt % FFKLVFF-PEG1k,
7.7 wt % FFKLVFF-PEG2k, and 20 wt % PEG10k-FFKLVFF.
The samples used to obtain these images were thick liquids/
thin gels.
The rheological response of fluids and gels was investigated for
FFKLVFF-PEG2k, through the study of the frequency response
of the dynamic shear moduli using controlled strain rheometry.
Figure 4 shows the data obtained for 2.5, 5, and 10 wt %
FFKLVFF-PEG2k.
(44) Teale, F. W. J.; Weber, G. Biochem. J. 1957, 65, 476–482.
Langmuir 2010, 26(12), 9986–9996
Castelletto et al.
Figure 3. Cryo-TEM images for (a) 1.8 wt % FFKLVFF-PEG1k,
(b) 1.5 wt % FFKLVFF-PEG2k, and (c) 1.9 wt % PEG10k-
FFKLVFF.
The modulus for 2.5 wt % sample is in the order of tens of
pascals (Figure 4a). For frequencies below ω = 10 rad s-1 there is
a terminal frequency scaling of approximately G0 , G00 ∼ ω1/3, while
there is a transition to a stronger frequency dependence of G0 ∼ ω1/2
at higher frequency that may reflect Rouse dynamics from
the PEG chains45 or may result from orientational ordering of
(45) Zanna, J. J.; Stein, P.; Marty, J. D.; Mauzac, M.; Martinoty, P. Macro-
molecules 2002, 35, 5459–5465.
(46) Shchipunov, Y. A.; Hoffmann, H. Langmuir 1998, 14, 6350–6360.
Langmuir 2010, 26(12), 9986–9996
Article
Figure 4. Dynamic shear moduli for FFKLVFF-PEG2k solu-
tions: (a) 2.5 wt % sample, 0.1% strain (preshear σ = 5 Pa for
1 min), (b) 5 wt % sample (preshear σ = 5 Pa for 1 min), and (c)
10 wt %, 0.1% strain (preshear σ = 10 Pa for 1 min).
rod-like micelles.46 The value of G0 for 5 wt % sample is on the
order of tens of pascals (Figure 4b). For frequencies lower than
ω = 130 rad s-1, there is a terminal frequency scaling G0 , G00 ∼
ω0.1, while for ω > 130 rad s-1, there is a transition to a stronger
frequency dependence of G0 ∼ ω0.8. The frequency response for
5 wt % FFKLVFF-PEG2k is similar to that measured previously
for a 5 wt % solution of FFKLVFF-PEG3k.23 The modulus is
about 1 order of magnitude lower, which may be due to the
difference in PEG chain length. A gel-like response is observed for
10 wt % FFKLVFF-PEG2k as shown in Figure 4c, both moduli
being nearly independent of frequency. The value of G0 is a little
lower than that previously measured for FFKLVFF-PEG3k;23
however, the shear thinning and recovery behavior (not shown)
were similar.
Flow alignment effects were investigated by SANS, which was
also used to identify liquid crystal phases in the higher concentra-
tion regime. During simultaneous SANS/shear flow experiments,
the shear rate γ_ was progressively increased from 0 to 5 s-1. All the
experiments have been performed in the radial configuration, i.e.,
the shear gradient-neutral plane is probed. Results from simulta-
neous shear flow/SANS experiments are shown in Figures 5, 6,
and Supporting Information Figure S3. The scattering patterns in
these figures correspond to the central scattering arising from
the peptide fibrils. In this work, we did not use an analytical
DOI: 10.1021/la100110f 9991
Article Castelletto et al.

Figure 5. SANS patterns for 6.9 wt % FFKLVFF-PEG1k. Bottom: one-dimensional profiles obtained from (a) a horizontal rectangular
sector, (b) a circular sector, and (c) a vertical rectangular sector integration. The SANS patterns correspond to the (a) as mounted sample or
sheared at (b) γ_ = 0.2 s-1 or (c) γ_ = 4 s-1.

expression to fit the peptide fibril form factor to the SANS data.
Instead, the SANS data were analyzed in terms of q*, which
defines the domain spacing d = (2π)/q*.
Figure 5a shows the SANS profile for 6.9 wt % FFKLVFF-
PEG1k as mounted in a Couette cell, while Figure 5b and c shows
the SANS data for the same sample sheared at γ_ = 0.2 and 4 s-1,
respectively. The value of q* = 0.035 Å-1 (for the sample as
mounted) indicates a domain spacing d = 180 Å, which is in good
agreement with the interfibrillar spacing from the cryo-TEM
image in Figure 3a.
Figure 5a shows that FFKLVFF-PEG1k fibrils are aligned
vertically upon mounting the sample in the Couette cell. The SANS
pattern became progressively isotropic for low shear rates
(Figure 5b). Increasing the shear rate leads to SANS patterns similar
to the one shown in Figure 5c. This shows alignment of fibrils along
the direction of the shear flow. SANS data in Figure 5, together with
the POM data (Supporting Information Figure S2), show that
FFKLVFF-PEG1k in a 6.9 wt % aqueous solution forms a nematic
liquid crystal phase. Supporting Information Figure S3a shows the
SANS profile for 7.7 wt % FFKLVFF-PEG2k as mounted in a
Couette cell, while Figure S3b,c shows the SANS data for the same
sample sheared at γ_ = 0.1 and 5 s-1, respectively. These data are
similar to that shown in Figure 5 for FFKLVFF-PEG1k. Figure 6. SANS patterns for 10.9 wt % FFYYKLVFF-PEG10k.
The SANS data in Supporting Information Figure S3a show Bottom: one-dimensional profiles obtained from (a) a circular
that the initial procedure of mounting the sample is enough to sector and (b) a vertical rectangular sector integration. The SANS
orient FFKLVFF-PEG2k fibrils. Figure S3b shows that, as soon patterns correspond to the (a) as mounted sample or sheared at (b)
γ_ = 4 s-1.
as the shear flow is started (γ_ = 0.1 s-1), the fibrils start to orient
in the direction of the shear flow. Increasing the shear rate enhan- Figure 6a shows the SANS data for 10.9 wt % PEG10k-
ces the degree of order of the fibers in the direction of the shear FFKLVFF as mounted in the Couette cell, while Figure 6b shows
flow (Figure S3c; γ_ = 5 s-1). The SANS patterns in Figure S3, the SANS data for the same sample sheared at γ_ = 4 s-1. This
together with the POM image (Figure S2b), show that 7.7 wt % sample did not attain any preferential orientation upon mounting
FFKLVFF-PEG2k forms a nematic liquid crystal phase in the sample in the Couette cell, as is shown by the isotropic pattern
aqueous solution. Furthermore, simultaneous shear flow/SANS in Figure 6a. However, upon increasing the shear flow, the fibrils
experiments revealed that SANS patterns become isotropic im- become oriented in the direction of the shear flow (Figure 6b, γ_ =
mediately after the shear is stopped. The domain spacing obtained 4 s-1). The SANS√pattern exhibits higher-order reflections in a
for this sample is similar to that for FFKLVFF-PEG1k (and positional ratio 1: 3:3 characteristic of a hexagonal columnar
PEG10k-FFKLVFF, vide infra) although the apparent fibril phase (Figure 6b). The value of q* = 0.03 Å-1 corresponds to a
spacing in the cryo-TEM image is larger. The origin of this domain spacing d = 209 Å. This is similar to the value for
apparent discrepancy is at present unclear. FFKLVFF-PEG1k, and reasonable in view of Figure 3c, although

9992 DOI: 10.1021/la100110f Langmuir 2010, 26(12), 9986–9996

Castelletto et al. Article

Figure 7. Raman spectra for a dried stalk prepared from a 10 wt %

PEG10k-FFKLVFF solution.

the fibrils are not as clearly resolved for PEG10k-FFKLVFF as for

the former sample.
The diffraction pattern of a randomly oriented hexagonal
columnar √ phase√ presents higher order reflections√ in a positional
order 1: 3:2: 7:3.47 The reflections at 2q* and 7q* are missing
in the pattern shown in Figure 6b, due to the orientation of the
sample under shear flow, and the influence of form factor which
leads to defined minima in the scattered intensity.
At this stage, it is necessary to point out that the extent of
anisotropy in the SANS pattern in Figure 5c and Supporting
Information Figure S3c is higher than that in Figure 6b due to the
different nature of the phases and their susceptibility to flow
alignment;the nematic phase is a fluid that can readily be aligned
by shear, whereas the hexagonal columnar phase is a soft gel that
cannot be oriented so easily.
In summary, POM revealed liquid crystal textures for solutions
of the three FFKLVFF-PEG conjugates. Rheology suggests
that the liquid crystal order might be present at concentrations as
low as 2.5 wt % for FFKLVFF-PEG2k. SANS experiments
confirmed nematic order for 6.9 wt % FFKLVFF-PEG1k,
7.7 wt % FFKLVFF-PEG2k, and hexagonal columnar order
for 10.9 wt % PEG10k-FFKLVFF.
Influence of PEG Crystallization. It was noted that, upon
drying, PEG crystallization could be observed using POM only
for PEG10k-FFKLVFF. Evidence for this is provided by a
characteristic spherulite structure observed by POM (Sup-
porting Information Figure S4). These spherulites are similar to
those observed for crystallizing polymers such as PEG and are not
of the same form as the amyloid “spherulites” observed pre-
viously, which exhibit a “maltese cross” pattern in the polarized
optical microscope.48,49
An ordered conformation of the PEG block in solution has Figure 8. Representative XRD patterns for (a) 6.9 wt % FFKLVFF-
already been discussed above in relation to FTIR results for PEG1k and (b) 10.9 wt % PEG10k-FFKLVFF.
PEG10k-FFKLVFF in solution (Figure 1d). The POM image in
Supporting Information Figure S4 shows that PEG10k block (Figure 1c). The region of the Raman spectra in Figure 7 with
fully crystallized in a dry film of PEG10k-FFKLVFF. wave numbers 1000-1500 cm-1 is very similar to the data
The influence of PEG crystallization, and its effect on peptide reported in the literature for crystallized PEG600.50 In particular,
β-sheet formation, was further characterized by Raman spectros- (A) is the region of the C-O vibration and (B) is the region of the
copy for PEG10k-FFKLVFF. Figure 7 shows the Raman spec- C-C vibration in the PEG chain.50
trum obtained for a dried stalk of 10 wt % PEG10k-FFKLVFF X-ray diffraction from a dried stalk was also used to investigate
solution. Raman bands at 1661 cm-1 and 1603 cm-1 in Figure 7 the morphology in the solid state and the influence of the PEG
are associated with antiparallel β-sheet structure,33 in good crystallization on the β-sheet structure of the samples. Figure 8
agreement with FTIR results for the same sample in solution contains some representative XRD patterns. Figure 8a,b contains
the data for stalks dried from FFKLVFF-PEG1k and PEG10k-
FFKLVFF solutions, respectively. A stalk dried from a FFKLVFF-
(47) International Tables for X-ray Crystallography; Kynoch Press: Birmingham,
1959; Vol. II. PEG2k solution was also studied by XRD (data not shown).
(48) Krebs, M. R. H.; MacPhee, C. E.; Miller, A. F.; Dunlop, L. E.; Dobson,
C. M.; Donald, A. M. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14420–14424.
(49) Krebs, M. R. H.; Bromley, E. H. C.; Rogers, S. S.; Donald, A. M. Biophys. (50) Kozielski, M.; Muhle, M.; Blaszczak, Z.; Szybowicz, M. Cryst. Res.
J. 2005, 88, 2013–2021. Technol. 2005, 4/5, 466–470.

Langmuir 2010, 26(12), 9986–9996 DOI: 10.1021/la100110f 9993

Article Castelletto et al.

Many of the reflections in Figure 8 can be indexed based on the

monoclinic unit cell of PEG (Supporting Information Table
ST1),51 proving that PEG1k and PEG10k crystallized when dried.
In addition, there is a “cross β” pattern with reflections at ∼11.5 Å
and ∼4.7 Å (which are superposed on a ring of scattering from
PEG). The observation of a cross β pattern indicates that at least
some β-sheet structure of FFKLVFF is retained upon PEG
crystallization, for the FFKLVFF/PEG conjugates studied in
this work. This result is in agreement with our previous work,26
where the retention of the cross β-sheet structure upon
PEG crystallization was proved for FFKLVFF-PEG3k. How-
ever, the reflection at ∼11.5 Å is much weaker for PEG10k-
FFKLVFF than it is for FFKLVFF-PEG1k and FFKLVFF-
PEG2K (data not shown), since the population of β sheets might
be reduced upon increasing the PEG length from PEG1k to
PEG10k.
Influence of Polyacrylic Acid: Stability of β-sheet Struc-
ture and Microphase Separation. It has recently been shown
that poly(acrylic acid) (PAA) can strongly associate with poly-
ethylene oxide (PEO) due to hydrogen bonding. The selective
association of PAA with PEO in PEO-PPO-PEO (“Pluronic”)
block copolymers led to microphase separation in the melt,28
which is not observed for Pluronic copolymers on their own
because the product χN (χ = Flory-Huggins interaction para-
meter, N = degree of polymerization) is not sufficiently large. The
observed phase separation for the PEO-PPO-PEO/PAA blends
results from an increase in χ. We attempted to increase the degree
of segregation between PEG and the FFKLVFF peptide in order
to produce microphase separated melts containing peptide do-
mains. As detailed below, microphase separation was in fact not
observed in the melt; however, the influence of PAA on the
crystallization of PEG and the adoption of a cross-β secondary
structure is explored. In fact, microphase separation between
PEG and the peptide is a natural consequence of the crystal-
lization of PEG in the solid state. Figure 9. DSC thermogram for (a) PEG10k-FFKLVFF heating
The microstructures of FFKLVFF/PEG conjugate/PAA ramp (2 °C/min) and (b) PAA20k heating ramp (10 °C/min).
blends were studied as a function of the PAA content and the also have hydrogen bonding interactions with the other com-
PEG or PAA molecular weights. A series of blends containing ponent.53
PAA20k or PAA88k and 100%, 75%, 50%, 25%, or 0% FFK- The change in slope of the heat flow profile in Figure 9b reveals
LVFF/PEG conjugate were prepared. The pure samples the glass transition of PAA20k at around 92 °C, which is similar
(PAA20k, PAA88k, FFKLVFF-PEG1k, FFKLVFF-PEG2k, to that reported in the literature.54,55
and PEG10k-FFKLVFF) were first studied by DSC to locate Some DSC experiments were also performed on FFKLVFF/
phase transitions associated with the glass transition (Tg) in PAA PEG conjugates/PAA melts. However, the formation of hydro-
and with melting/crystallization of PEG in the FFKLVFF/PEG gen bonds between the PAA and the conjugates resulted in broad
conjugates. Then, both the pure samples and the blends were melting ranges in the DSC thermograms, making the interpreta-
studied by XRD. tion of the DSC data for the blends difficult.
Representative DSC traces for PEG10k-FFKLVFF and PAA20k Figure 10 shows the XRD profiles corresponding to the
are shown in Figure 9a,b, respectively. This data shows that the FFKLVFF-PEG1k/PAA20k and FFKLVFF-PEG1k/PAA88k
melting point of PEG10k block in the conjugate is 59.7 °C, blends as a function of the FFKLVFF/PEG conjugate content.
slightly lower (3.8 °C) than that of pure PEG10k reported in the The indexation of the reflections in Figure 10 is shown in
literature at a similar heating rate.52 The lower melting point of Supporting Information Table ST2. The results do not show
the PEG10k block in the conjugate may originate from the microphase separation, but PEG crystallization and β-sheet
hydrogen bonding between the amide group of the peptide formation, as will be detailed below.
and the ether linkage of the PEG segment. Such competitive The data in Figure 10 indicate similar features for the two
hydrogen bonding interactions weaken the β-sheet formation of different PAA samples (Supporting Information Table ST2). The
the peptides. The hydrogen bonding interaction between XRD profiles denote PEG crystallization and a population of
FFKLVFF and the PEG block may affect the chain-folding FFKLVFF β-sheets for blends containing 25% PAA20k or
and packing of the PEG block, hence reducing the crystallinity PAA80k. PEG does not crystallize for PAA contents equal or
of PEG block and its melting point. It is similar to the
phenomenon observed for the melting point depression in
(53) Painter, P. C.; Shenoy, S. L.; Bhagwagar, D. E.; Fishburn, J.; Coleman,
polymer blends where one component can crystallize and M. M. Macromolecules 1991, 24, 5623–5629.
(54) Cascone, M. G.; Polacco, G.; Lazzeri, L.; Barbani, N. A. J. Appl. Polym.
(51) Takahashi, Y.; Tadokoro, H. Macromolecules 1973, 6, 672–675. Sci. 1997, 66, 2089–2094.
(52) Pielichowski, K.; Flejtuch, K. Polym. Adv. Technol. 2002, 13, 690–696. (55) Chu, C.-H.; Berner, B. J. Appl. Polym. Sci. 1993, 47, 1083–1087.

9994 DOI: 10.1021/la100110f Langmuir 2010, 26(12), 9986–9996

Castelletto et al.
Figure 10. Representative XRD profiles for FFKLVFF/PEG
conjugate blends with (a) PAA20k and (b) PAA88k as a function
of the peptide content.
higher than 50% PAA20k or 50% PAA88k. A population of
FFKLVFF cross β-sheets can be clearly observed for blends
containing up to 75% PAA20k or 75% PAA88k. However, the
stacking of the β-sheets in the direction perpendicular to the plane
of the strands seems to be disrupted for 50% and 75% PAA20k or
PAA88k. This is denoted by a splitting of the XRD peak centered
at ∼11 Å into two peaks centered at ∼10 and ∼12 Å.
Separate synchrotron small-angle X-ray scattering data, along
with the data in Figure 10 (which cover a range up to higher q)
indicates there is no microphase separation in the FFKLVFF-
PEG1k blends.
Results similar to those described above (not reported in this
paper) have been found for blends of FFKLVFF-PEG2k or
PEG10k-FFKLVFF with PAA20k or PAA80k.
Conclusions
We report the self-assembly behavior of PEGylated FFKLVFF
conjugates, containing PEG1k, PEG2k, and PEG10k. Our results
show that FFKLVFF-PEG conjugates are model peptide/PEG
systems that form core-shell fibrils and, at higher concentration,
lyotropic liquid crystal phases.
FTIR, CD, and cryo-TEM results show that FFKLVFF/PEG
conjugates start to self-assemble into fibrils, with a FFKLVFF
β-sheet core and a PEG corona, at 1 wt % concentration in
aqueous solution. The formation of aggregates in solution was also
verified at low concentrations ([0.005-0.06] wt % FFKLVFF/
PEG conjugate) using fluorescence and CD, such that these
aggregates are characterized by associations between phenylala-
nine units. This result repeats the same self-assembly process
already determined by us for a wide family of PEGylated peptide
conjugates,23,29,41 i.e., an initial formation of aggregates with
hydrophobic interactions at low concentrations, followed at higher
concentration by self-assembly into β-sheets.
In comparison with our earlier work on FFKLVFF-PEG3k,23,24
the CD and FTIR data presented here provide information on the
Langmuir 2010, 26(12), 9986–9996
Article
influence of PEG molar mass on the secondary structure. It seems
that the extent of β-sheet ordering is reduced in PEG10k-
FFKLVFF compared to the other two samples, reflecting the
increased influence of the highest molar mass PEG studied. This
may be due to the effect of the attached PEG on the ordering of the
peptide residues, in particular, those constrained by location close to
the junction point with the polymer.43 Cryo-TEM shows a decrease
in the resolution of the fibrils for PEG10k-FFKLVFF compared to
the other two, again reflecting the influence of PEG, since in this
case, the higher density peptide core comprises a lower fraction of
the fibril mass.
POM, together with SANS, confirmed nematic order for
6.9 wt % FFKLVFF-PEG1k and 7.7 wt % FFKLVFF-PEG2k.
Hexagonal order was verified by SANS for 10.9 wt % PEG10k-
FFKLVFF. In particular, additional rheology experiments
proved that the liquid crystal phase of FFKLVFF-PEG1k might
be present for concentrations as low as 2.5 wt %. These results
may be compared to our previous observations of nematic and
hexagonal columnar mesophase formation of FFKLVFF-
PEG3k23,24 where these phases were observed successively on
increasing concentration. As discussed elsewhere,23,24,56 the for-
mation of these phases can be rationalized on the basis of theories
for the packing of semiflexible chains. The fact that nematic
ordering is observed indicates that the fibril length (persistence
length) to diameter ratio is large enough and the observation of
hexagonal columnar ordering indicates that the polydispersity in
fibril diameter is sufficiently low.
PEG crystallization is observed by FTIR for FFKLVFF-
PEG2k and PEG10k-FFKLVFF in solution. Similarly, Raman
and XRD indicated PEG crystallization upon drying solutions of
FFKLVFF/PEG conjugates containing PEG1k, PEG2k, or
PEG10k. In our work, PEG crystallization was always observed
along with the FFKLVFF cross β structure. It is therefore evident
that the β-sheet structure is not perturbed by PEG crystallization
for the FFKLVFF/PEG conjugates containing PEG1k, PEG2k,
or PEG10k.
In keeping with our previous results,23,24 FFKLVFF/PEG
conjugates with PEG1k, PEG2k, PEG3k, and PEG10k have a
strong fibrillization tendency, consistent with the number of
aromatic residues in the sequence. These results support our
conclusions for several related peptide/PEG conjugates concern-
ing the influence of PEG crystallization.26,27 We showed that
strong fibrillizing peptides such as YYKLVFF retain β-sheet
secondary structure when PEG crystallizes,29 although this is
disrupted for more weakly fibrillizing peptides such as KLVFF.
The study of FFKLVFF-PEG/PAA blends helped us to
qualitatively evaluate the stability of the FFKLVFF β-sheet
structure and PEG crystallization. While PEG did not crystallize
for PAA blend contents equal to or higher than 50%, the
FFKLVFF β-sheet structure was still observed for blends con-
taining 75% PAA.
Our results provide information on the stability of the hydro-
phobic peptide structure, in self-assembled fibrils in solution and
against PEG crystallization. Formation of microphase separated
structures in the melt was not observed, even when adding a
hydrogen-bonding polymer in an attempt to increase incompat-
ibility between synthetic and peptide components. The control of
crystallization, and hence ultimate properties, in blends with PAA
may offer potential benefits in applications of biomaterials, where
these are present in the solid form, for instance, in substrates for
sensors or supports.
(56) Hamley, I. W. Soft Matter 2010, in press.
DOI: 10.1021/la100110f 9995
Article
Acknowledgment. This work was supported by EPSRC grants
EP/F048114/1 and EP/G026203/1 to IWH. We are grateful
to Steve Furzeland and Derek Atkins (Unilever, Colworth,
UK) for performing the cryo-TEM experiments. We would like
to acknowledge Dr. Rebecca Green (Dept. of Pharmacy,
Univ. of Reading) for access to the FTIR instrument and
9996 DOI: 10.1021/la100110f
Castelletto et al.
Mr. Nick Spencer (Biocentre, Univ. of Reading) for assistance
with XRD experiments.
Supporting Information Available: Additional graphics
and data as described in the text. This material is available
free of charge via the Internet at http://pubs.acs.org.
Langmuir 2010, 26(12), 9986–9996