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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 time.1,7,12-17 A nice review discusses applications of PEGylation
because it is possible to combine synergistically the properties of of peptides and proteins for applications in biotechnology.18
peptides (functionality, responsiveness) with those of synthetic From a more fundamental viewpoint, it is of interest to examine
polymers (solubility, responsiveness, cheapness).1-8 Short pep- whether the secondary structure of the polypeptide is influenced
tides attached to polymers have been shown to be capable of when linked to PEG. From another perspective, these conjugates
guiding the polymer superstructure.6-8 This could become a may exhibit amphiphilic behavior similar to that of conventional
powerful method to control polymer morphology and properties, surfactants. Nonionic surfactants often contain poly(ethylene glycol)
using biologically inspired end/side groups. From the other (PEG) conjugated to a short hydrophobic moiety (commonly an
perspective, polymers may be used as supports to present peptide alkyl chain with six to eighteen carbon atoms).
functionality within polymeric matrices (gels) or to guide the The influence of polymer chain length on the self-assembly of
ordering of peptide units via amphiphilic-type self-assembly. polymer/peptide conjugates has previously been investigated by
Several recent reviews focus on polymer/peptide conjugates and several groups. Klok and co-workers have investigated the self-
discuss different approaches to their synthesis using convergent assembly of PEG-peptides containing bioinspired coiled coil
and divergent methods, based on growth of polymer from tethered peptide sequences.19,20 The conjugates prepared had PEG molec-
peptides, or vice versa, or coupling of presynthesized units.3,5,9-12 ular weights of either 750 g mol-1 or 2000 g mol-1. The focus was
Conjugation of PEG to peptides and proteins is of great on the stabilization of the coiled coil peptide structure against pH,
interest because PEG provides a neutral, water-soluble polymeric concentration, and temperature changes conferred by PEG. In
coating around the biomolecule that can reduce uptake of the general, little effect of PEG molar mass was noted when pH was
varied. In the case of varying peptide concentration, the higher
(1) L€owik, D. W. P. M.; Ayres, L.; Smeenk, J. M.; van Hest, J. C. M. Adv.
Polym. Sci. 2006, 202, 19–52.
(2) Heredia, K. L.; Maynard, H. D. Org. Biomol. Chem. 2007, 5, 45–53. (13) Delgado, C.; Francis, G. E.; Fisher, D. Crit. Rev. Ther. Drug 1992, 9, 249–
(3) Van Hest, J. C. M. J. Macromol. Sci. C 2007, 47, 63. 304.
(4) B€orner, H. G.; Schlaad, H. Soft Matter 2007, 3, 394–408. (14) Zalipsky, S. Adv. Drug Deliver. Rev. 1995, 16, 157–182.
(5) Gauthier, M. A.; Klok, H.-A. Chem. Commun. 2008, 2591–2611. (15) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40, 539–
(6) B€orner, H. G. Prog. Polym. Sci. 2009, 34, 811–851. 551.
(7) Klok, H.-A. J. Polym. Sci.: Polym. Chem. 2005, 43, 1–17. (16) Veronese, F. M. Biomaterials 2001, 22, 405–417.
(8) B€orner, H. G.; Kuehnle, H.; Hentschel, J. J. Polym. Sci.: Polym. Chem. 2010, (17) B€orner, H. G.; Smarsly, B.; Hentschel, J.; Rank, A.; Schubert, R.; Geng, Y.;
48, 1–14. Discher, D. E.; Hellweg, T.; Brandt, A. Macromolecules 2008, 41, 1430–1437.
(9) Nicolas, J.; Mantovani, G.; Haddleton, D. M. Macromol. Rapid Commun. (18) Ryan, S. M.; Mantovani, G.; Wang, X.; Haddleton, D. M.; Brayden, D. J.
2007, 28, 1083–1111. Expert Opin. Drug Delivery 2008, 5, 371–383.
(10) Koning, H. M.; Kilbinger, A. F. M. Angew. Chem., Int. Ed. Engl. 2007, 46, (19) Vandermeulen, G. W. M.; Tziatzios, C.; Duncan, R.; Klok, H.-A. Macro-
8334–8340. molecules 2005, 38, 761–769.
(11) Marsden, H. R.; Kros, A. Macromol. Biosci. 2009, 9, 939–951. (20) Klok, H.-A.; Vandermeulen, G. W. M.; Nuhn, H.; Rosler, A.; Hamley,
(12) Lutz, J. F.; B€orner, H. G. Prog. Polym. Sci. 2008, 33, 1–39. 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. Article

molar mass PEG led to a lower helix content20 or to enhanced Experimental Section
stability against pH change in the case of a heteropeptide Materials. The FFKLVFF/PEG conjugates FFKLVFF-
conjugate containing oppositely charged residues designed to PEG1k and FFKLVFF-PEG2k were synthesized by Rapp poly-
favor electrostatic interpolyelectrolyte complex formation.19 mere GmbH (T€ ubingen, Germany) using solid-phase peptide
B€orner and co-workers have investigated polymer-peptide con- synthesis methods, and were supplied as HCl salts. The synthesis
jugates based on poly(n-butyl acrylate) (PnBA) of varying mo- method is similar to that used to prepare YYKLVFF-PEG
lecular weight conjugated to a β-sheet peptide (TV)5 aggregating polymers already reported by us.29
domain peptide.4 The dimensions of fibrillar aggregates imaged The conjugates FFKLVFF-PEG1K and FFKLVFF-PEG2K
by AFM were found to increase with PnBA chain length, and the were characterized (by the supplier) by reverse-phase high-per-
kinetics of self-assembly in solution were found to be retarded for formance liquid chromatography (RP-HPLC; Grom Saphir 200,
C18 5 μm column). A mobile phase of a gradient of water with
the highest molar mass sample (PnBA 38k). Lynn and co-workers
0.1% TFA and acetonitrile with 0.75% TFA was used to confirm
have investigated the self-assembly of conjugates of PEG with an high purity. Sample elution was monitored using a UV/vis
amyloid β (Aβ) peptide fragment, Aβ(10-35)-PEG3k,21 but did detector operating at 220 nm.
not examine the influence of PEG molar mass. Conjugation to MALDI-TOF (Ultraflex, Bruker with matrix Universalmatrix,
PEG was found to enhance the solubility of the Aβ peptide, and Fluka) as performed by the supplier was used to confirm Mw =
led to reversible fibrillization, in contrast to the native peptide. 1895 g mol-1 for FFKLVFF-PEG1k, while GPC provided Mn =
We have recently investigated the self-assembly of PEG/pep- 1046.8 g mol-1 for the precursor PEG. MALDI-TOF confirmed
tides containing peptides based on the sequence KLVFF, Aβ- Mw = 3095 g mol-1 (Mw/Mn < 1.05) for FFKLVFF-PEG2k and
(17-20), from the amyloid beta (Aβ) peptide. This core sequence Mn = 2103 g mol-1 for the precursor PEG.
has been shown to be important in fibrillization.22 We have PEG10k-FFKLVFF was obtained from American Peptide
Inc. (Sunnyvale, USA) as a TFA salt. The peptide fragment (Mpr-
investigated the self-assembly in aqueous solution of FFKLVFF-
Phe-Phe-Lys-Leu-Val-Phe-Phe) was synthesized on Fmoc-Phe-
PEG3k (PEG3k denotes PEG with approximate Mn = 3000 g mol-1) Wang resin by using standard Fmoc/tBu chemistry. Protecting
and found that it forms lyotropic liquid crystal phases at groups used for amino acids are as follows: Trt group for Mpr and
sufficiently high concentration in water.23-25 We have also Boc for Lys. Fmoc-protected amino acids were purchased from
shown that PEG3k crystallizes when FFKLVFF/PEG3k con- EMD Biosciences and GL Biochem. Reagents for coupling and
jugates are dried at room temperature, leading to an interplay cleavage were purchased from Aldrich. Solvents were purchased
between PEG crystallization and peptide fibrillization.26,27 In from Fisher Scientific. The peptide chain was assembled on resin
the present paper, the influence of PEG chain length and by repetitive removal of the Fmoc protecting group by treating
position is investigated, using again the model FFKLVFF with 20% piperidine/DMF for 30 min and coupling of protected
sequence used in our previous work. The peptide FFKLVFF amino acid with HBTU/HOBt/NMM for 90 min. Ninhydrin
testing was performed after each coupling to check the coupling
is itself hydrophobic, indeed does not dissolve in water; however,
efficiency. After the last coupling, the resulting resin was washed
when conjugated to PEG it provides a suitable hydrophile/ and dried, then treated with reagent K (TFA/thioanisole/phenol/
lipophile balance such that FFKLVFF-PEG conjugates show EDT/water, 87.5:5:2.5:2.5:2.5, v/v) 3 h for cleavage and removal
excellent amphiphilic properties. Here, we study conjugates with of the side chain protecting groups. Crude peptide was precipi-
PEG1k or PEG2k attached at the C terminus as in our previous tated from cold ether and collected by filtration. The peptide
study of PEG3k, and also PEG10k, which for synthetic reasons fragment was purified by reverse-phase HPLC to 95%, pooled
was attached at the N terminus of the peptide. We show that fractions were lyophilized to dry. Yield was 108 mg. MS of the
these samples also exhibit lyotropic liquid crystallinity; in peptide before PEG10k conjugation provided Mw = 1035.3 g mol-1.
particular, nematic and hexagonal columnar phases are ob- The above peptide substrate (108 mg, 0.1 mmol) was then
served depending on concentration and PEG chain length. We conjugated with 1 equiv PEG10K-Maleimide (1000 mg, 0.1 mmol)
in aqueous solution at pH 8. GPC provided Mn = 9531 g mol-1
also investigate the influence of PEG molar mass on the
and Mw = 9872 g mol-1 (PDI 1.04) for the precursor PEG10k.
morphology in dried samples;only PEG of sufficient molecular The PEGylated product was further purified by reversed-phase
weight is capable of crystallizing. Finally, the influence of HPLC to 95%, and the pooled fractions were lyophilized to dry.
addition of an associating polymer, poly(acrylic acid) (PAA), Poly(acrylic acid) PAA20 (Mw = 21 800 g mol-1, Mn = 20 000 g
which can form a hydrogen-bonded complex with PEG,28 was mol-1) and PAA88 (Mw = 98 500 g mol-1, Mn = 88 000 g mol-1)
examined. This was motivated by an attempt to increase the were purchased from Polymer Source Inc. (Quebec, Canada) and
degree of segregation between the PEG and peptide domains, used as received.
with the objective of producing microphase separation in the Peptide solutions were prepared by mixing weighed amounts of
melt state. This was not achieved under the conditions studied; peptide and Milli-Q water and allowing the sample to mix by
nonetheless, the effect of PAA on the structure in the solid state diffusion over a period of ∼7 days. Peptide/polymer blends were
made by codissolving them at a given weight ratio in water. After
was probed. Specifically, the influence of PAA on PEG crystal-
spontaneous solvent evaporation, the blends were annealed at
lization and cross-β fibril structure was examined as a function 80 °C for 24 h. Allowing for solvent evaporation and the
of blend composition. subsequent annealing at 80 °C provided thin films of blends, used
for XRD studies.
(21) Burkoth, T. S.; Benzinger, T. L. S.; Jones, D. N. M.; Hallenga, K.; Fourier Transform Infrared (FTIR) Spectroscopy. Spec-
Meredith, S. C.; Lynn, D. G. J. Am. Chem. Soc. 1998, 120, 7655–7656. tra were measured on a Nicolet Nexus spectrometer with DTGS
(22) Hamley, I. W. Angew. Chem., Int. Ed. Engl. 2007, 46, 8128–8147. detector. Solutions of FFKLVFF-PEG1k, FFKLVFF-PEG2k,
(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. and PEG10k-FFKLVFF in D2O (0.9, 2, 3.2, 3.6, 5, 6.9, 7.7, 11.3,
(24) Hamley, I. W.; Krysmann, M. J.; Castelletto, V.; Noirez, L. Adv. Mater. and 20 wt %) were sandwiched between two CaF2 plate windows
2008, 20, 4394–4397. (spacer 0.006 mm). Spectra were scanned 128 times over the range
(25) Hamley, I. W.; Krysmann, M. J.; Newby, G. E.; Castelletto, V.; Noirez, L. 4000-900 cm-1.
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. (29) Castelletto, V.; Newby, G. E.; Hermida-Merino, D.; Hamley, I. W.; Liu, D.;
(28) Tirumala, V. R.; Ilavsky, J.; Ilavsky, M. J. Chem. Phys. 2006, 124, 234911. Noirez, L. Polym. Chem. 2009, in press.

Langmuir 2010, 26(12), 9986–9996 DOI: 10.1021/la100110f 9987

Article Castelletto et al.

Raman Spectroscopy. Raman spectra were recorded using a X-ray Diffraction (XRD). The experiments were performed
Renishaw inVia Raman microscope. The light source was a using a RAXIS IVþþ X-ray diffractometer (Rigaku) equipped
multiline laser, such that the experiments were performed using with a rotating anode generator. The XRD data was collected
the λ = 785 nm edge. Experiments were made on stalks prepared using a Saturn 992 CCD camera. Diffraction patterns for the
by drying filaments of the peptide obtained from a 10 wt % pure peptides were obtained for stalks prepared by drying fila-
PEG10k-FFKLVFF sample. The stalks were focused by using a ments of the peptide. Aqueous solutions of FFKLVFF-PEG1k
20
magnification lens. Spectra were obtained in the interval (6.9 wt %), FFKLVFF-PEG2k (7.7 wt %), and PEG10k-
(100-3000) cm-1, using 20 s collection time with 10% laser power FFKLVFF (10.9 wt %) were suspended between the ends of a
and taking two averages. wax-coated capillary and dried. Thin films of blends contain-
Circular Dichroism (CD). Spectra were recorded using a ing the FFKLVFF/PEG conjugate and (0-100) % PAA20k or
Chirascan spectropolarimeter (Applied Photophysics, UK). CD (0-100) % PAA88k were also studied by XRD. The stalks or the
was performed using FFKLVFF-PEG1k, FFKLVFF-PEG2k, thin films were mounted (vertically) onto the four axis goniometer
or PEG10k-FFKLVFF dissolved in water (0.05, 0.06, 0.9, and of the X-ray diffractometer.
1 wt %) and loaded into quartz coverslip cuvettes (0.1-mm-thick) Rheology. Rheological properties were determined using a
or into 1-mm-thick quartz bottles. Spectra are presented with controlled-stress TA Instruments AR-2000 rheometer (TA In-
absorbance A < 2 at any measured point with a 0.5 nm step, 1 nm struments). For a fluid 5 wt % solution of FFKLVFF-PEG2k, a
bandwidth, and 1 s collection time per step at 20 °C. Mooney geometry was used. For a gel-like 10 wt % sample, a
Fluorescence Spectroscopy. Spectra were recorded using a cone-and-plate geometry (cone diameter 20 mm, angle 1°) was
Cary Eclipse Varian Fluorescence Spectrometer with samples in a used. Frequency sweeps were performed at 25 °C. Preliminary
1.0 cm quartz cuvette. Spectra were measured for samples in strain sweeps were performed for each sample in order to define
water (0.005 or 0.007 wt %). The spectra were recorded from 279 the linear viscoelastic region, thus ensuring that moduli were
to 490 nm using an excitation wavelength λex = 265 nm. independent of strain.
Cryogenic-Transmission Electron Microscopy (Cryo-TEM). Differential Scanning Calorimetry (DSC). Melting points
Experiments were performed at Unilever Research, Colworth for the FFKLVFF/PEG conjugates and the glass transition
(Bedford, UK). Solutions of FFKLVFF-PEG1k, FFKLVFF- temperature of the PAA were measured by DSC using a Mettler
PEG2k, or PEG10k-FFKLVFF (1.5, 1.8, 1.9 wt %) were blotted DSC 823 system, at heating rates of 2 °C/min and 10 °C/min,
and vitrified using a Gatan Cp3 cryoplunge system. Samples were respectively.
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 Results
a 400-mesh copper TEM grid (Agar) covered with a per-
forated carbon film (plasma-treated). The drop was automatically Self-Assembly in Solution and Secondary Structure of
blotted, and the sample was plunged into liquid ethane (-183 °C) FFKLVFF/PEG Conjugates. An important question is whether
to form a vitrified specimen,30,31 then transferred to liquid nitro- the secondary structure of the peptide is retained in the PEG/
gen (-196 °C) for storage. Specimens were examined in a JEOL peptide conjugate. This was investigated by FTIR and CD
JEM-2100 electron microscope at 200 kV, at temperatures below spectroscopy.
-175 °C. Images were recorded digitally on a Gatan UltraScan The secondary structure of the self-assembled peptides in
1000 cooled CCD camera using DigitalMicrograph (Gatan) in the solution was first studied by FT-IR in the transmission config-
low-dose imaging mode to minimize beam exposure and electron- uration. Figure 1a-c show the Amide I and Amide II regions of
beam radiation damage. the FTIR spectra measured for D2O solutions of the three
Small-Angle Neutron Scattering (SANS). Small-angle FFKLVFF/PEG conjugates.
neutron scattering (SANS and rheo-SANS) was performed on
The FTIR spectra for (0.9-6.9) wt % FFKLVFF-PEG1k
6.9 wt % FFKLVFF-PEG1k, 7.7 wt % FFLVFF-PEG2k, and
10.9 wt % PEG10k-FFKLVFF samples, using the 2D sensitive
(Figure1a) exhibit maxima at 1682 and 1618 cm-1. The max-
multidetector PAXY of the Laboratoire L
eon Brillouin. Samples imum at 1618 cm-1 becomes more intense upon increasing
were placed in a quartz Couette cell (0.1 mm gap) which was used concentration, compared to the peak at 1682 cm-1. Figure 1b
to apply steady shear.32 Shear rates are defined as γ_ = ΩRh/(R0 - shows that, while the FTIR spectra for 0.9 wt % FFKLVFF-
R1), where Ω is the angular velocity, Rh is the average radius, and PEG2k shows only one well-defined peak at 1617 cm-1, the
R0 = 19.0 mm and R1 = 19.1 mm are the inner and outer radii. FTIR spectra for 3.6 and 7.7 FFKLVFF-PEG2k present three
Shear rates applied were γ_ = 0.1-5 s-1. Measurements were per- maxima at 1682, 1672, and 1617 cm -1.
formed at room temperature. Data were obtained with neutrons Figure 1c shows that the FTIR spectra for 2-20 wt % PEG10k-
incident along the shear gradient direction (radial configuration). FFKLVFF present two maxima at 1700 and 1674 cm-1 and a third
A wavelength of 6 Å was used. The sample-detector distance maximum which shifts from 1625 to 1631 cm-1 upon increasing
was fixed at 2.5 m. The corresponding q range extends from 0.023 concentration The intensity of the peak at (1625-1631) cm-1
to 0.129 Å-1. increases upon increasing the concentration, while the remaining
Polarized Optical Microscopy (POM). Microscopy experi- two peaks are nearly insensitive to the concentration. It also is
ments were performed by placing the sample between crossed noticeable that a peak at 1553 cm-1 starts to develop for 11.3 wt %
polarizers in an Olympus BX41 polarized microscope. FKLVFF-
peptide and becomes clear in the spectrum for 20 wt % peptide.
PEG1k (6.9 wt %), FFKLVFF-PEG2K (7.8 wt %), and PEG10k-
FFKLVFF (20 wt %) samples were placed between a glass slide The FTIR spectra in Figure 1a-c provide comparative in-
and a coverslip before capturing the images with a Canon G2 formation about the secondary structure of the FFKLVFF/PEG
digital camera. A few drops of 11.5 wt % PEG10k-FFKLVFF conjugates, as a function of the PEG length and the sample
were also placed on a glass slide and left to dry before capturing the concentration.
image with a Canon G2 digital camera. FTIR peaks at (1617-1631) cm-1 are associated with a β-sheet
structure.33,34 The simultaneous presence of peaks at (1617-1631)
(30) Talmon, Y. (1999) Cryogenic transmission electron microscopy in the study of cm-1 and at (1682 -1700) cm-1 suggests an antiparallel β-sheet
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.; (33) Haris, P.; Chapman, D. Biopolymers 1995, 37, 251–263.
Lubovsky, M.; Talmon, Y.; Pochan, D. J. Soft Matter 2007, 3, 945–955. (34) Stuart, B. (1997) Biological Applications of Infrared Spectroscopy; Wiley:
(32) Baroni, P.; Pujolle, C.; Noirez, L. Rev. Sci. Instrum. 2001, 72, 2686. Chichester.

9988 DOI: 10.1021/la100110f Langmuir 2010, 26(12), 9986–9996

Castelletto et al. Article

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 is shown in Figure 1d for 11.3 wt % PEG10k-FFKLVFF. The
contain contributions from TFA, corresponding to the peak at spectrum contains peaks at 1035, 1090, 1139, 1254, 1332, and
(1672-1674) cm-1 (Figure 1b,c). The TFA content in FFKLVFF- 1350 cm-1 which are usually associated with semicrystalline
PEG2k solutions might consist of residual solvent from the HPLC PEG.39 These results indicate that the length of the PEG block
process, while the PEG10k-FFKLVFF was provided as a TFA salt in FFKLVFF-PEG2k and PEG10k-FFKLVFF allows for its
by the manufacturer. The peak at 1533 cm-1 measured in Figure 1c ordering in solution, but does not disrupt the stability of the
is associated with the amide II band,34,37 and arises from the β-sheet structure.
N-H in-plane bending or C-N stretching modes of the amide The crystallization of the PEG block will be addressed later
backbone.38 in this work regarding samples dried from solutions of the
According to our previous studies,23,24 FFKLVFF-PEG3k FFKLVFF/PEG conjugates.
self-assembles into fibrils in aqueous solution at concentrations CD was used to probe changes in the secondary structure of the
similar to those studied in Figure 1. The fibrils consist of a peptide self-assembly, as a function of concentration and the PEG
hydrophobic core containing the FFKLVFF block, surrounded block length. Figure 2a shows the CD spectra obtained for
by a hydrophilic corona containing the PEG block. The FFKLVFF solutions containing (0.05-0.06) wt % FFKLVFF/PEG conju-
block is arranged in β-sheet strands within the fibril hydrophobic gates, while Figure 2b contains CD results for samples containing
core. 1 wt % FFKLVFF/PEG conjugates.
In good agreement with previous data for FFKLVFF-PEG3k The CD spectra for the dilute solutions in Figure 2a are domi-
fibrils,23,24 the FTIR results in Figure 1 show the existence of nated by a maximum at ∼218 nm and a minimum at ∼232 nm.
antiparallel β-sheet structure in solutions of FFKLVFF-PEG1k, A prominent single maximum at ∼220 nm and a minimum at
FFKLVFF-PEG2k, and PEG10k-FFKLVFF, such that the 230 nm have been previously reported by us in the CD spectrum
population of β-sheets increases upon increasing the concentra- for FFKLVFF-PEG3k,23 FFKLVFF,40 and FFFF-PEG3k.41
tion of the sample, at least for the former two samples. The spectra According to our previous work, and in agreement with CD
for PEG10k-FFKLVFF are more complex, which may reflect an results in the literature for phenylalanine oligopeptides,42 a strong
increased influence of the PEG chain on the ordering of the positive peak at 218 nm may result from the π-π* stacking. On
peptide as discussed below in the context of results from CD the other hand, spectra resembling those in Figure 2 reported for
spectroscopy. peptide amphiphiles lacking aromatic residues have also been
Features in the FTIR spectra in the region 1370-1000 cm-1 ascribed to a coexistence of β-sheet ordering of peptide at the core
can provide information about PEG crystallization. Indeed, only of the fibril with polyproline II type ordering of strands at the
the FTIR spectra for the FFKLVFF/PEG conjugates containing periphery.43 A complex variation in the conformation of the
PEG2k and PEG10k show some features corresponding to the
ordering of the PEG block in solution. A representative example (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–
(35) Rosler, A.; Klok, H.-A.; Hamley, I. W.; Castelletto, V.; Mykhaylyk, O. O. 1406.
Biomacromolecules 2003, 4, 859–863. (41) Castelletto, V.; Hamley, I. W. Biophys. Chem. 2009, 141, 169–174.
(36) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712–719. (42) Peggion, E.; Palumbo, M.; Bonora, G. M.; Toniolo, C. Bioorg. Chem. 1974,
(37) Lin, S.-Y.; Chu, H.-L. Int. J. Biol. Macromol. 2003, 32, 173–177. 3, 125.
(38) Sarkar, S.; Chourasia, A.; Maji, S.; Sadhukran, S.; Kumar, S.; Adhikari, B. (43) Paramonov, S. E.; Jun, H. W.; Hartgerink, J. D. J. Am. Chem. Soc. 2006,
Mater. Sci. 2006, 29, 475–484. 128, 7291–7298.

Langmuir 2010, 26(12), 9986–9996 DOI: 10.1021/la100110f 9989

Article 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
Figure 2. Molar ellipticity for solutions of (a) (0.05-0.06) wt % contrast between core and shell was observed, indicating that the
and (b) (0.9-1) wt % FFKLVFF/PEG conjugates. core (and possibly adjacent high density inner shell region) only
are imaged.
strand as a function of distance from the junction point may The length of the fibers in Figure 3 is rather polydisperse,
likewise be anticipated in our PEG/peptide conjugates. extending up to several micrometers (based on these images,
The inset in Figure 2a shows the ellipticity values at ∼218 nm and several others obtained for these samples). It has to be
(region A) and at ∼232 nm (region B) as a function of the PEG pointed out that, for a similar concentration of FFKLVFF/
length. The ellipticity at 232 nm is weakly sensitive to the PEG PEG conjugate, the number density of long fibres in the cryo-
length, while the ellipticity at 218 increases with PEG length. It is TEM images decreases upon increasing the PEG length. It is
possible that this indicates a decrease in β-sheet ordering upon possible that excluded volume interactions between PEG cor-
increasing the PEG chain length. This is not unreasonable onas of neighboring fibril, increase the distance between fibrils
considering that a lengthy PEG chain may be expected to more upon increasing the PEG length, hence reducing the number
strongly influence the packing, in this case disrupting the β-sheet density of fibrils.
hydrogen bonding, of more of the residues in the peptide. In summary, FTIR and cryo-TEM results indicate that
Figure 2b shows that for 1 wt % FFKLVFF/PEG conjugates the FFKLVFF/PEG conjugates self-assemble into fibrils with a
there is an evolution in the CD spectra as a function of the PEG β-sheet structure at concentrations as low as 1 wt % FFKLVFF/
block length. The CD spectrum for 0.9 wt % FFKLVFF-PEG1k PEG conjugate. CD and fluorescence results suggest the forma-
has negative bands centered at 215 and 222 nm. The minimum at tion of aggregates in solution for (0.005-0.06) wt % FFKLVFF/
215 nm is associated with a β-sheet structure. The β-sheet PEG conjugate. Although our results do not provide evidence
minimum becomes gradually canceled by a positive band which about the geometry of the self-assembled structure of the aggre-
starts to grow at 219 nm in the CD spectra, upon increasing the gates at low concentration, it is certain that interactions between
PEG length from PEG1k to PEG10k (Figure 2b). The minimum neighboring phenylalanine units play an important role in their
at 222 nm observed for PEG1k gradually shifts to 226 and 230 nm structure.
for PEG2k and PEG10k, respectively. Liquid Crystal Phase Formation and Dynamics of Shear
The CD signal for samples containing PEG1k in Figure 2b is Flow Alignment for FFKLVFF/PEG Conjugates. Liquid
dominated by the β-sheet structure of the fibrils, in good agree- crystal phase formation was noted at higher concentration, via
ment with FTIR results in Figure 1a. For longer PEG chains, the characteristic birefringence textures observed by polarized optical
growth of a positive band at 219 nm reflects the increased microscopy. Figure S2 (Supporting Information) shows the
influence of PEG on residues close to the junction point for birefringence textures obtained for 6.9 wt % FFKLVFF-PEG1k,
which the β-sheet ordering is disrupted, possibly replaced by 7.7 wt % FFKLVFF-PEG2k, and 20 wt % PEG10k-FFKLVFF.
polyproline II ordering, as discussed by Paramonov for peptide The samples used to obtain these images were thick liquids/
amphiphiles (in which the core comprises hydrophobic lipid and thin gels.
the shell is peptide, i.e., the inverse structure to our system).43 The rheological response of fluids and gels was investigated for
These observations may shed light on the complex FTIR spectra FFKLVFF-PEG2k, through the study of the frequency response
shown in Figure 1, although analysis of these in terms of the of the dynamic shear moduli using controlled strain rheometry.
variable ordering of residues as a function of distance from the Figure 4 shows the data obtained for 2.5, 5, and 10 wt %
PEG junction point is complex. FFKLVFF-PEG2k.
The inset in Figure 2b shows the ellipticity values in regions (A)
and (B) of the CD spectra increase with PEG length. Phenylalanine (44) Teale, F. W. J.; Weber, G. Biochem. J. 1957, 65, 476–482.

9990 DOI: 10.1021/la100110f Langmuir 2010, 26(12), 9986–9996

Castelletto et al. 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
Figure 3. Cryo-TEM images for (a) 1.8 wt % FFKLVFF-PEG1k,
(b) 1.5 wt % FFKLVFF-PEG2k, and (c) 1.9 wt % PEG10k-
lower than that previously measured for FFKLVFF-PEG3k;23
FFKLVFF. however, the shear thinning and recovery behavior (not shown)
were similar.
The modulus for 2.5 wt % sample is in the order of tens of Flow alignment effects were investigated by SANS, which was
pascals (Figure 4a). For frequencies below ω = 10 rad s-1 there is also used to identify liquid crystal phases in the higher concentra-
a terminal frequency scaling of approximately G0 , G00 ∼ ω1/3, while tion regime. During simultaneous SANS/shear flow experiments,
there is a transition to a stronger frequency dependence of G0 ∼ ω1/2 the shear rate γ_ was progressively increased from 0 to 5 s-1. All the
at higher frequency that may reflect Rouse dynamics from experiments have been performed in the radial configuration, i.e.,
the PEG chains45 or may result from orientational ordering of 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
(45) Zanna, J. J.; Stein, P.; Marty, J. D.; Mauzac, M.; Martinoty, P. Macro-
molecules 2002, 35, 5459–5465. these figures correspond to the central scattering arising from
(46) Shchipunov, Y. A.; Hoffmann, H. Langmuir 1998, 14, 6350–6360. the peptide fibrils. In this work, we did not use an analytical

Langmuir 2010, 26(12), 9986–9996 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. 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
Figure 10. Representative XRD profiles for FFKLVFF/PEG hexagonal columnar ordering indicates that the polydispersity in
conjugate blends with (a) PAA20k and (b) PAA88k as a function fibril diameter is sufficiently low.
of the peptide content. PEG crystallization is observed by FTIR for FFKLVFF-
PEG2k and PEG10k-FFKLVFF in solution. Similarly, Raman
higher than 50% PAA20k or 50% PAA88k. A population of and XRD indicated PEG crystallization upon drying solutions of
FFKLVFF cross β-sheets can be clearly observed for blends
FFKLVFF/PEG conjugates containing PEG1k, PEG2k, or
containing up to 75% PAA20k or 75% PAA88k. However, the
PEG10k. In our work, PEG crystallization was always observed
stacking of the β-sheets in the direction perpendicular to the plane
along with the FFKLVFF cross β structure. It is therefore evident
of the strands seems to be disrupted for 50% and 75% PAA20k or
that the β-sheet structure is not perturbed by PEG crystallization
PAA88k. This is denoted by a splitting of the XRD peak centered
for the FFKLVFF/PEG conjugates containing PEG1k, PEG2k,
at ∼11 Å into two peaks centered at ∼10 and ∼12 Å.
Separate synchrotron small-angle X-ray scattering data, along or PEG10k.
with the data in Figure 10 (which cover a range up to higher q) In keeping with our previous results,23,24 FFKLVFF/PEG
indicates there is no microphase separation in the FFKLVFF- conjugates with PEG1k, PEG2k, PEG3k, and PEG10k have a
PEG1k blends. strong fibrillization tendency, consistent with the number of
Results similar to those described above (not reported in this aromatic residues in the sequence. These results support our
paper) have been found for blends of FFKLVFF-PEG2k or conclusions for several related peptide/PEG conjugates concern-
PEG10k-FFKLVFF with PAA20k or PAA80k. ing the influence of PEG crystallization.26,27 We showed that
strong fibrillizing peptides such as YYKLVFF retain β-sheet
Conclusions secondary structure when PEG crystallizes,29 although this is
disrupted for more weakly fibrillizing peptides such as KLVFF.
We report the self-assembly behavior of PEGylated FFKLVFF The study of FFKLVFF-PEG/PAA blends helped us to
conjugates, containing PEG1k, PEG2k, and PEG10k. Our results qualitatively evaluate the stability of the FFKLVFF β-sheet
show that FFKLVFF-PEG conjugates are model peptide/PEG structure and PEG crystallization. While PEG did not crystallize
systems that form core-shell fibrils and, at higher concentration, for PAA blend contents equal to or higher than 50%, the
lyotropic liquid crystal phases. FFKLVFF β-sheet structure was still observed for blends con-
FTIR, CD, and cryo-TEM results show that FFKLVFF/PEG taining 75% PAA.
conjugates start to self-assemble into fibrils, with a FFKLVFF Our results provide information on the stability of the hydro-
β-sheet core and a PEG corona, at 1 wt % concentration in phobic peptide structure, in self-assembled fibrils in solution and
aqueous solution. The formation of aggregates in solution was also against PEG crystallization. Formation of microphase separated
verified at low concentrations ([0.005-0.06] wt % FFKLVFF/ structures in the melt was not observed, even when adding a
PEG conjugate) using fluorescence and CD, such that these hydrogen-bonding polymer in an attempt to increase incompat-
aggregates are characterized by associations between phenylala- ibility between synthetic and peptide components. The control of
nine units. This result repeats the same self-assembly process crystallization, and hence ultimate properties, in blends with PAA
already determined by us for a wide family of PEGylated peptide may offer potential benefits in applications of biomaterials, where
conjugates,23,29,41 i.e., an initial formation of aggregates with these are present in the solid form, for instance, in substrates for
hydrophobic interactions at low concentrations, followed at higher
sensors or supports.
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 (56) Hamley, I. W. Soft Matter 2010, in press.

Langmuir 2010, 26(12), 9986–9996 DOI: 10.1021/la100110f 9995

Article Castelletto et al.

Acknowledgment. This work was supported by EPSRC grants Mr. Nick Spencer (Biocentre, Univ. of Reading) for assistance
EP/F048114/1 and EP/G026203/1 to IWH. We are grateful with XRD experiments.
to Steve Furzeland and Derek Atkins (Unilever, Colworth,
UK) for performing the cryo-TEM experiments. We would like Supporting Information Available: Additional graphics
to acknowledge Dr. Rebecca Green (Dept. of Pharmacy, and data as described in the text. This material is available
Univ. of Reading) for access to the FTIR instrument and free of charge via the Internet at http://pubs.acs.org.

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