Self-Assembly of PEGylated Peptide Conjugates Containing a

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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 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 peptides 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. (2) Heredia, K. L.; Maynard, H. D. Org. Biomol. Chem. 2007, 5, 45–53. (3) Van Hest, J. C. M. J. Macromol. Sci. C 2007, 47, 63. (4) B€orner, H. G.; Schlaad, H. Soft Matter 2007, 3, 394–408. (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. (9) Nicolas, J.; Mantovani, G.; Haddleton, D. M. Macromol. Rapid Commun. 2007, 28, 1083–1111. (10) Koning, H. M.; Kilbinger, A. F. M. Angew. Chem., Int. Ed. Engl. 2007, 46, 8334–8340. (11) Marsden, H. R.; Kros, A. Macromol. Biosci. 2009, 9, 939–951. (12) Lutz, J. F.; B€orner, H. G. Prog. Polym. Sci. 2008, 33, 1–39.

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conjugate and can enhance in vivo circulation or residence 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 selfassembly of PEG-peptides containing bioinspired coiled coil peptide sequences.19,20 The conjugates prepared had PEG molecular 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. (15) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 2001, 40, 539– 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. Macromolecules 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.

Published on Web 05/07/2010

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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 conjugates based on poly(n-butyl acrylate) (PnBA) of varying molecular 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/peptides 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 FFKLVFFPEG3k (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 conjugates 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 observed 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 crystallization 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.

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Experimental Section Materials. The FFKLVFF/PEG conjugates FFKLVFFPEG1k and FFKLVFF-PEG2k were synthesized by Rapp polymere 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-performance 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 (MprPhe-Phe-Lys-Leu-Val-Phe-Phe) was synthesized on Fmoc-PheWang 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 precipitated 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. Spectra 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.

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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, FFKLVFFPEG2k, 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 perforated 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 nitrogen (-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 electronbeam 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 Leon 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 performed at room temperature. Data were obtained with neutrons incident along the shear gradient direction (radial configuration). A wavelength of 6 A˚ was used. The sample-detector distance was fixed at 2.5 m. The corresponding q range extends from 0.023 to 0.129 A˚-1. Polarized Optical Microscopy (POM). Microscopy experiments were performed by placing the sample between crossed polarizers in an Olympus BX41 polarized microscope. FKLVFFPEG1k (6.9 wt %), FFKLVFF-PEG2K (7.8 wt %), and PEG10kFFKLVFF (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.

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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 filaments of the peptide. Aqueous solutions of FFKLVFF-PEG1k (6.9 wt %), FFKLVFF-PEG2k (7.7 wt %), and PEG10kFFKLVFF (10.9 wt %) were suspended between the ends of a wax-coated capillary and dried. Thin films of blends containing 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 Instruments). 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 configuration. 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 maximum 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 % FFKLVFFPEG2k 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 % PEG10kFFKLVFF 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 information 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.

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Figure 1. Amide I/II regions of FTIR spectra for samples in D2O at the concentrations indicated: (a) FFKLVFF-PEG1k, (b) FFKLVFFPEG2k, 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 FFKLVFFPEG2k 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 concentration 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.

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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 conjugates, 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 dominated 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.

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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 agreement 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

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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 minimum corresponding to the n-π* transition in the CD spectra. Fluorescence spectroscopy confirmed the role of aromatic stacking interactions between phenylalanine residues in the selfassembly 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 cryoTEM. Cryo-TEM was performed instead of conventional negative 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 FFKLVFFPEG1k, FFKLVFF-PEG2k, and PEG10k-FFKLVFF, respectively. 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 cryoTEM images decreases upon increasing the PEG length. It is possible that excluded volume interactions between PEG coronas 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 formation 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 aggregates 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.

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Figure 4. Dynamic shear moduli for FFKLVFF-PEG2k solutions: (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).

Figure 3. Cryo-TEM images for (a) 1.8 wt % FFKLVFF-PEG1k, (b) 1.5 wt % FFKLVFF-PEG2k, and (c) 1.9 wt % PEG10kFFKLVFF.

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. Macromolecules 2002, 35, 5459–5465. (46) Shchipunov, Y. A.; Hoffmann, H. Langmuir 1998, 14, 6350–6360.

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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 concentration 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 simultaneous 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

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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 % FFKLVFFPEG1k 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 A˚-1 (for the sample as mounted) indicates a domain spacing d = 180 A˚, 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. The SANS data in Supporting Information Figure S3a show that the initial procedure of mounting the sample is enough to orient FFKLVFF-PEG2k fibrils. Figure S3b shows that, as soon 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 enhances the degree of order of the fibers in the direction of the shear flow (Figure S3c; γ_ = 5 s-1). The SANS patterns in Figure S3, together with the POM image (Figure S2b), show that 7.7 wt % FFKLVFF-PEG2k forms a nematic liquid crystal phase in aqueous solution. Furthermore, simultaneous shear flow/SANS experiments revealed that SANS patterns become isotropic immediately after the shear is stopped. The domain spacing obtained for this sample is similar to that for FFKLVFF-PEG1k (and PEG10k-FFKLVFF, vide infra) although the apparent fibril spacing in the cryo-TEM image is larger. The origin of this apparent discrepancy is at present unclear. 9992 DOI: 10.1021/la100110f

Figure 6. SANS patterns for 10.9 wt % FFYYKLVFF-PEG10k. Bottom: one-dimensional profiles obtained from (a) a circular sector and (b) a vertical rectangular sector integration. The SANS patterns correspond to the (a) as mounted sample or sheared at (b) γ_ = 4 s-1.

Figure 6a shows the SANS data for 10.9 wt % PEG10kFFKLVFF as mounted in the Couette cell, while Figure 6b shows the SANS data for the same sample sheared at γ_ = 4 s-1. This sample did not attain any preferential orientation upon mounting the sample in the Couette cell, as is shown by the isotropic pattern in Figure 6a. However, upon increasing the shear flow, the fibrils become oriented in the direction of the shear flow (Figure 6b, γ_ = 4 s-1). The SANS√pattern exhibits higher-order reflections in a positional ratio 1: 3:3 characteristic of a hexagonal columnar phase (Figure 6b). The value of q* = 0.03 A˚-1 corresponds to a domain spacing d = 209 A˚. This is similar to the value for FFKLVFF-PEG1k, and reasonable in view of Figure 3c, although Langmuir 2010, 26(12), 9986–9996

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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 √ in a positional √ phase √ presents higher order reflections 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 (Supporting 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 previously, which exhibit a “maltese cross” pattern in the polarized optical microscope.48,49 An ordered conformation of the PEG block in solution has already been discussed above in relation to FTIR results for PEG10k-FFKLVFF in solution (Figure 1d). The POM image in Supporting Information Figure S4 shows that PEG10k block fully crystallized in a dry film of PEG10k-FFKLVFF. The influence of PEG crystallization, and its effect on peptide β-sheet formation, was further characterized by Raman spectroscopy for PEG10k-FFKLVFF. Figure 7 shows the Raman spectrum obtained for a dried stalk of 10 wt % PEG10k-FFKLVFF solution. Raman bands at 1661 cm-1 and 1603 cm-1 in Figure 7 are associated with antiparallel β-sheet structure,33 in good agreement with FTIR results for the same sample in solution (47) International Tables for X-ray Crystallography; Kynoch Press: Birmingham, 1959; Vol. II. (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. J. 2005, 88, 2013–2021.

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Figure 8. Representative XRD patterns for (a) 6.9 wt % FFKLVFFPEG1k and (b) 10.9 wt % PEG10k-FFKLVFF.

(Figure 1c). The region of the Raman spectra in Figure 7 with wave numbers 1000-1500 cm-1 is very similar to the data reported in the literature for crystallized PEG600.50 In particular, (A) is the region of the C-O vibration and (B) is the region of the C-C vibration in the PEG chain.50 X-ray diffraction from a dried stalk was also used to investigate the morphology in the solid state and the influence of the PEG crystallization on the β-sheet structure of the samples. Figure 8 contains some representative XRD patterns. Figure 8a,b contains the data for stalks dried from FFKLVFF-PEG1k and PEG10kFFKLVFF solutions, respectively. A stalk dried from a FFKLVFFPEG2k solution was also studied by XRD (data not shown). (50) Kozielski, M.; Muhle, M.; Blaszczak, Z.; Szybowicz, M. Cryst. Res. Technol. 2005, 4/5, 466–470.

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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 A˚ and ∼4.7 A˚ (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. However, the reflection at ∼11.5 A˚ is much weaker for PEG10kFFKLVFF than it is for FFKLVFF-PEG1k and FFKLVFFPEG2K (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 Structure and Microphase Separation. It has recently been shown that poly(acrylic acid) (PAA) can strongly associate with polyethylene 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 parameter, 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 domains. 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 crystallization of PEG in the solid state. The microstructures of FFKLVFF/PEG conjugate/PAA blends were studied as a function of the PAA content and the PEG or PAA molecular weights. A series of blends containing PAA20k or PAA88k and 100%, 75%, 50%, 25%, or 0% FFKLVFF/PEG conjugate were prepared. The pure samples (PAA20k, PAA88k, FFKLVFF-PEG1k, FFKLVFF-PEG2k, and PEG10k-FFKLVFF) were first studied by DSC to locate phase transitions associated with the glass transition (Tg) in PAA and with melting/crystallization of PEG in the FFKLVFF/PEG conjugates. Then, both the pure samples and the blends were studied by XRD. Representative DSC traces for PEG10k-FFKLVFF and PAA20k are shown in Figure 9a,b, respectively. This data shows that the melting point of PEG10k block in the conjugate is 59.7 °C, slightly lower (3.8 °C) than that of pure PEG10k reported in the literature at a similar heating rate.52 The lower melting point of the PEG10k block in the conjugate may originate from the hydrogen bonding between the amide group of the peptide and the ether linkage of the PEG segment. Such competitive hydrogen bonding interactions weaken the β-sheet formation of the peptides. The hydrogen bonding interaction between FFKLVFF and the PEG block may affect the chain-folding and packing of the PEG block, hence reducing the crystallinity of PEG block and its melting point. It is similar to the phenomenon observed for the melting point depression in polymer blends where one component can crystallize and (51) Takahashi, Y.; Tadokoro, H. Macromolecules 1973, 6, 672–675. (52) Pielichowski, K.; Flejtuch, K. Polym. Adv. Technol. 2002, 13, 690–696.

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Figure 9. DSC thermogram for (a) PEG10k-FFKLVFF heating ramp (2 °C/min) and (b) PAA20k heating ramp (10 °C/min).

also have hydrogen bonding interactions with the other component.53 The change in slope of the heat flow profile in Figure 9b reveals the glass transition of PAA20k at around 92 °C, which is similar to that reported in the literature.54,55 Some DSC experiments were also performed on FFKLVFF/ PEG conjugates/PAA melts. However, the formation of hydrogen bonds between the PAA and the conjugates resulted in broad melting ranges in the DSC thermograms, making the interpretation of the DSC data for the blends difficult. Figure 10 shows the XRD profiles corresponding to the FFKLVFF-PEG1k/PAA20k and FFKLVFF-PEG1k/PAA88k blends as a function of the FFKLVFF/PEG conjugate content. The indexation of the reflections in Figure 10 is shown in Supporting Information Table ST2. The results do not show microphase separation, but PEG crystallization and β-sheet formation, as will be detailed below. The data in Figure 10 indicate similar features for the two different PAA samples (Supporting Information Table ST2). The XRD profiles denote PEG crystallization and a population of FFKLVFF β-sheets for blends containing 25% PAA20k or PAA80k. PEG does not crystallize for PAA contents equal or (53) Painter, P. C.; Shenoy, S. L.; Bhagwagar, D. E.; Fishburn, J.; Coleman, M. M. Macromolecules 1991, 24, 5623–5629. (54) Cascone, M. G.; Polacco, G.; Lazzeri, L.; Barbani, N. A. J. Appl. Polym. Sci. 1997, 66, 2089–2094. (55) Chu, C.-H.; Berner, B. J. Appl. Polym. Sci. 1993, 47, 1083–1087.

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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 A˚ into two peaks centered at ∼10 and ∼12 A˚. 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 FFKLVFFPEG1k 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 phenylalanine 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

influence of PEG molar mass on the secondary structure. It seems that the extent of β-sheet ordering is reduced in PEG10kFFKLVFF 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 % PEG10kFFKLVFF. 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 FFKLVFFPEG3k23,24 where these phases were observed successively on increasing concentration. As discussed elsewhere,23,24,56 the formation 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 FFKLVFFPEG2k 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 concerning 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 containing 75% PAA. Our results provide information on the stability of the hydrophobic 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 incompatibility 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.

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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

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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.

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