Templating Silica Nanostructures on Rationally Designed Self

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Templating Silica Nanostructures on Rationally Designed Self-Assembled Peptide Fibers Stewart C. Holmstro¨m,† Patrick J. S. King,† Maxim G. Ryadnov,†,| Michael F. Butler,‡ Stephen Mann,*,† and Derek N. Woolfson*,†,§ School of Chemistry, UniVersity of Bristol, Bristol BS8 1TS, U.K., UnileVer Research, Colworth, Bedfordshire, MK44 1LQ, U.K., and Department of Biochemistry, UniVersity of Bristol, Bristol BS8 1TD, U.K. ReceiVed June 26, 2008. ReVised Manuscript ReceiVed August 6, 2008 Nature presents exquisite examples of templating hard, functional inorganic materials on soft, self-assembled organic substrates. An ability to mimic and control similar processes in the laboratory would increase our understanding of fundamental science, and may lead to potential applications in the broad arena of bionanotechnology. Here we describe how self-assembled, R-helix-based peptide fibers of de novo design can promote and direct the deposition of silica from silicic acid solutions. The peptide substrate can be removed readily through proteolysis, or other facile means to render silica nanotubes. Furthermore, the resulting silica structures, which span the nanometer to micrometer range, can themselves be used to template the deposition of the cationic polyelectrolyte, poly-(diallyldimethylammonium chloride). Finally, the peptide-based substrates can be engineered prior to silicification to alter the morphology and mechanical properties of the resulting hybrid and tubular materials.

Introduction Inorganic oxide nanotubes have recently gained much attention as novel materials for emerging technologies in areas such as electronics, photonics, nanofluidics, sensing, catalysis and controlled release.1 Nanotubes can be prepared by templatedirected methods using a wide range of materials such as nanorods,2 electrospun polymer fibers,3 nanoporous membranes,4 DNA,5 phospholipid cylinders,6,7 organic gel filaments,8 needlelike organic crystals,9 viroid cylinders,10 collagen fibers,11 porphyrin nanotapes,12 and most recently peptide amphiphiles.13 Many of these examples utilize sol-gel reactions to prepare nanotubes based on amorphous silica. In this regard, recent studies have investigated the mechanisms responsible for the promotion of organosilane hydrolysis and polysilicate condensation in the presence of various organic macromolecules14 such as synthetic polymers,15 homopeptides,16,17 polyamines18-20 peptide mim* To whom correspondence should be addressed. E-mail: s.mann@ bristol.ac.uk (S.M.); [email protected] (D.N.W.). † School of Chemistry, University of Bristol. ‡ Unilever Research. § Department of Biochemistry, University of Bristol. | Present address: Department of Chemistry, University of Leicester, Leicester, LE1 7RH, U.K. (1) Xia, Y. N.; Yang, P. D.; Sun, Y. G.; Wu, Y. Y.; Mayers, B.; Gates, B.; Yin, Y. D.; Kim, F.; Yan, Y. Q. AdV. Mater. 2003, 15, 353–389. (2) Lauhon, L. J.; Gudiksen, M. S.; Wang, C. L.; Lieber, C. M. Nature 2002, 420, 57–61. (3) Peng, Q.; Sun, X.-Y.; Spagnola, J. C.; Hyde, K. G.; Spontak, R. J.; Parsons, G. N. Nano Lett. 2007, 7, 719–722. (4) Martin, C. R. Science 1994, 266, 1961–1966. (5) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775– 778. (6) Baral, S.; Schoen, P. Chem. Mater. 1993, 5, 145–147. (7) Patil, A. J.; Muthusamy, E.; Seddon, A. M.; Mann, S. AdV. Mater. 2003, 15, 1816–1819. (8) Ono, Y.; Nakashima, K.; Sano, M.; Kanekiyo, Y.; Inoue, K.; Hojo, J.; Shinkai, S. Chem. Commun. 1998, 14, 1477–1478. (9) Miyaji, F.; Davis, S. A.; Charmant, J. P. H.; Mann, S. Chem. Mater. 1999, 11, 3021–3024. (10) Shenton, W.; Douglas, T.; Young, M.; Stubbs, G.; Mann, S. AdV. Mater. 1999, 11, 253–256. (11) Ono, Y.; Kanekiyo, Y.; Inoue, K.; Hojo, J.; Nango, M.; Shinkai, S. Chem. Lett. 1999, 6, 475–476. (12) Meadows, P. J.; Dujardin, E.; Hall, S. R.; Mann, S. Chem. Commun. 2005, 29, 3688–3690. (13) Yuwono, V. M.; Hartgerink, J. D. Langmuir 2007, 23, 5033–5038.

ics21,22 and peptide-polymer hybrids.23 In several of these cases, regions of positive charge, or, purportedly hydroxyl moieties, along the polymer chain appear to be responsible for the induction of silica deposition from aqueous solution.19,24,25 Many of these examples have been inspired by studies on proteins such as silaffins,26 and silicateins,27-29 which are extracted from silica biominerals associated with diatoms and sponges,30 respectively. These proteins catalyze the in vitro precipitation of nanostructured silica when added to silicic acid solutions under ambient conditions. For example, in model studies at neutral pH, addition of such proteinaceous bioextracts leads to an approximately 10-fold increase in the yield of silica, compared to experiments undertaken in the presence of denatured silicatein or control proteins such as bovine serum albumin and trypsin.31 More specifically, a 19-amino acid peptide fragment, R5 (SSKKSGSYSGSKGSKRRIL), obtained from silaffin isolated from Cylindrotheca fusiformis, is reported to be highly efficient at inducing and regulating silica precipitation when exposed to (14) Patwardhan, S. V.; Clarson, S. J.; Perry, C. C. Chem. Commun. 2005, 9, 1113–1121. (15) Patwardhan, S. V.; Clarson, S. J. Silicon Chem. 2002, 1, 207–241. (16) Coradin, T.; Livage, J. Colloids Surf., B 2001, 21, 329–336. (17) Patwardhan, S. V.; Mukherjee, N.; Clarson, S. J. J. Inorg. Organomet. Polym. 2001, 11, 193–198. (18) Kroger, N.; Deutzmann, R.; Bergsdorf, C.; Sumper, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14133–14138. (19) Mizutani, T.; Nagase, H.; Fujiwara, N.; Ogoshi, H. Bull. Chem. Soc. Jpn. 1998, 71, 2017–2022. (20) Sumper, M. Science 2002, 295, 2430–2433. (21) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature 2000, 403, 289–292. (22) Patwardhan, S. V.; Clarson, S. J. Polym. Bull. 2002, 48, 367–371. (23) Kessel, S.; Thomas, A.; Borner, H. G. Angew. Chem., Int. Ed. 2007, 46, 9023–9026. (24) Coradin, T.; Durupthy, O.; Livage, J. Langmuir 2002, 18, 2331–2336. (25) Sudheendra, L.; Raju, A. R. Mater. Res. Bull. 2002, 37, 151–159. (26) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129–1132. (27) Cha, J. N.; Shimizu, K.; Zhou, Y.; Christiansen, S. C.; Chmelka, B. F.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 361–365. (28) Shimizu, K.; Cha, J.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6234–6238. (29) Tacke, R. Angew. Chem., Int. Ed. 1999, 38, 3015–3018. (30) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. (31) Perry, C. C.; Keeling-Tucker, T. Colloid Polym. Sci. 2003, 281, 652–664.

10.1021/la802009t CCC: $40.75  2008 American Chemical Society Published on Web 08/30/2008

Designed Protein Fibers Template Silica

a silane precursor under ambient conditions.26,32,33 Notably, negligible silica is formed when the RRIL motif of R5 is removed.34 Though post-translational modification of lysinelysine clusters in silaffins have been reputed to play a central role in silica formation under acidic conditions,35 recent work using a synthetic R5 peptide indicates that these modifications are not necessary for silica precipitation under neutral or slightly basic conditions.36 However, the direct role of such arginine-containing repeats in these proteins has been questioned very recently37s Wenzl and co-workers provide evidence that similar RRL repeats in a new class of proteins, called the silacidins, act as linkers between otherwise polyanionic peptides. They propose that the linkers are removed to render completely anionic chains, which can bind polyamines. It is these complexes that are proposed to template silica. Whatever the final details, the above examples indicate that silica deposition from aqueous solution depends on the chemical functionality of soluble macromolecules. This suggests that nanoscale objects with appropriate surface moieties may serve as effective templates for the high-fidelity transcription of silica replicas. In this regard, the ability to design the self-assembly and surface chemistry of peptide nanofilaments and fibers would provide a rational and wide-ranging approach to new types of organic templates with high anisotropy and biocompatibility for silica deposition and bioinorganic composite fabrication. The use of fibrillar peptide-based structures as templates has been previously reported. For example, in a series of studies, Matsui with co-workers demonstrated that metallic nanocrystals can be grown on the surfaces of nanotubes assembled from mineralization-promoting peptide sequences38-42 In addition, hollow silica nanotubes can be prepared by sol-gel condensation of tetraethoxysilane (TEOS) specifically onto a self-assembling β-sheet peptidesan amyloid-like assemblysfollowed by degradation of the organic core at high temperatures (calcinations).43 Similarly, Yuwono and Hartgerink recently describe templating of silica by cationic peptide amphiphiles, which are β-sheet forming peptides with alkyl chains conjugated at the N-terminus; this method also employs TEOS, with the organic component being removed by calcination.13 Here we present methods for preparing silica nanotubes at ambient temperatures and physiological pH, which combines the use of self-assembled R-helical fibers of de novo design (SAFs) as the template and silicic acid as the silica-forming agent. We demonstrate that the peptide template can be removed in a facile manner by employing a protease to render nanotubes, and that the key morphological principles underlying SAF (32) Lenoci, L.; Camp, P. J. J. Am. Chem. Soc. 2006, 128, 10111–10117. (33) Sumper, M.; Kroger, N. J. Mater. Chem. 2004, 14, 2059–2065. (34) Knecht, M. R.; Wright, D. W. Chem. Commun. 2003, 24, 3038–3039. (35) Kroger, N.; Deutzmann, R.; Sumper, M. J. Biol. Chem. 2001, 276, 26066– 26070. (36) Brott, L. L.; Naik, R. R.; Pikas, D. J.; Kirkpatrick, S. M.; Tomlin, D. W.; Whitlock, P. W.; Clarson, S. J.; Stone, M. O. Nature 2001, 413, 291–293. (37) Wenzl, S.; Hett, R.; P., R.; Sumper, M. Angew. Chem., Int. Ed. 2008, 47, 1–5. (38) Banerjee, I. A.; Yu, L. T.; Matsui, H. Proc. Nat. Acad. Sci. U.S.A. 2003, 100, 14678–14682. (39) Djalali, R.; Chen, Y. F.; Matsui, H. J. Am. Chem. Soc. 2003, 125, 5873– 5879. (40) Yu, L. T.; Banerjee, I. A.; Matsui, H. J. Am. Chem. Soc. 2003, 125, 14837–14840. (41) Yu, L. T.; Banerjee, I. A.; Matsui, H. J. Mater. Chem. 2004, 14, 739–743. (42) Yu, L. T.; Banerjee, I. A.; Shima, M.; Rajan, K.; Matsui, H. AdV. Mater. 2004, 16, 709. (43) Meegan, J. E.; Aggeli, A.; Boden, N.; Brydson, R.; Brown, A. P.; Carrick, L.; Brough, A. R.; Hussain, A.; Ansell, R. J. AdV. Funct. Mater. 2004, 14, 31–37.

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assembly44,45 can be applied to the fabrication of shape-controlled nanotubes. The SAF system comprises two R-helical peptidessSAF-p1 and SAF-p2asdesigned to coassemble into a staggered, or sticky ended R-helical heterodimer.44,46,47 The sticky ends are complementary and, thus, the dimer presents a building block for fibrillogenesis. The resulting matured fibers are 50-100 nm thick and tens of micrometers long. Compared with the aforementioned amyloid-like systems, the SAF system is relatively straightforward to engineer further. Specifically, we have succeeded in introducing branches, kinks and functional entities into the otherwise straight fibers.44,45,48 SAFs possess a net positive charge at neutral pH, which could favor the system as a template for silica deposition.13 Moreover, the SAF sequences show similarities to the purported silica-nucleating peptides discussed above: they are enriched in cationic amino acids (K and R), and these are interspaced with the hydroxy amino acids (S and Y), which may also play a role in silicification.49 On the basis of these structural and sequence features, we tested for silica deposition on the surfaces of the SAFs. Herein, we demonstrate that silica-peptide hybrid fibers can be generated using the SAF template at near-neutral pH via a facile approach based on previous work carried out within the Mann group.50 The resulting silicified structures have peptide cores encased in silica shells of controllable thickness. These hybrid structures can be functionalized further by coating with an ultrathin layer of the cationic polyelectrolyte, poly-(diallyldimethylammonium chloride) (PDADMAC). The peptide cores can be removed either by exposure to salt, calcination, extensive exposure to water or proteolysis to produce hollow nanotubes of silica. Finally, changes in SAF morphology, introduced by the presence of specials, are transcribed into nanoscale silica replicas. To our knowledge, this is the first example of silica deposition onto a designed nanoscale R-helix-based fibrous template with integrated design features to promote biomineralization under benign conditions.

Experimental Methods Peptide Templates. SAF peptides and orthogonal conjugates B2C and C2N were prepared using standard automated solid-phase methods and Fmoc chemistry as previously described.44,45 The SAF peptide sequences are KIAALKQKIASLKQEIDALEYENDALEQ, SAF-p1, and KIRRLKQKNARLKQEIAALEYEIAALEQ, SAFp2a. Each has two distinct halves: SAF-p1 comprises A (KIAALKQKIASLKQ) and B (EIDALEYENDALEQ); and SAF-p2a C (KIRRLKQKNARLKQ) and D (EIAALEYEIAALEQ); where A complements D and B complements C in peptide self-assembly. Fiber samples (20 µL) were prepared to give final concentrations of 100 µM in each peptide, by mixing aqueous solutions of SAF peptides in MOPS buffer (10 mM, 0.22 µm filtered, pH 7.4) and incubating overnight at 20 °C. Similar procedures were used to prepare SAFs with nonlinear morphologies; specials were mixed with standard SAF peptides in ratios of 1:1:0.01 (p1/p2a/C2N) and 1:1:0.1 (p1/p2a/B2C). Silicic Acid (Si(OH)4) Preparation. Silicic acid solutions (20 mM) were prepared according to methods described previously.51 (44) Ryadnov, M. G.; Woolfson, D. N. Nat. Mater. 2003, 2, 329–332. (45) Ryadnov, M. G.; Woolfson, D. N. J. Am. Chem. Soc. 2005, 127, 12407– 12415. (46) Pandya, M. J.; Spooner, G. M.; Sunde, M.; Thorpe, J. R.; Rodger, A.; Woolfson, D. N. Biochemistry 2000, 39, 8728–8734. (47) Smith, A. M.; Banwell, E. F.; Edwards, W. R.; Pandya, M. J.; Woolfson, D. N. AdV. Funct. Mater. 2006, 16, 1022–1030. (48) Ryadnov, M. G.; Woolfson, D. N. Angew. Chem., Int. Ed. 2003, 42, 3021–3023. (49) Hecky, R. E.; Mopper, K.; Kilham, P.; Degens, E. T. Mar. Biol. 1973, 19, 323–331. (50) Hall, S. R.; Bolger, H.; Mann, S. Chem. Commun. 2003, 22, 2784–2785. (51) Hall, S. R.; Davis, S. A.; Mann, S. Langmuir 2000, 16, 1454–1456.

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Figure 1. TEM micrographs illustrating the synthesis of silica nanotubes on peptide templates with various morphologies. Key: column 1, selfassembled peptide scaffolds formed by mixing specified peptides and overnight incubation at 20 °C. Column 2, silicified peptide scaffolds. Column 3, silica replicas left after proteolysis of the protein cores with chymotrypsin. Row 1, standard linear SAFs. Row 2, segmented morphologies. Row 3, kinked morphologies. Scale bars: column 1, 1 µm; columns 2 and 3, 200 nm. Note: one structure in panel H appears to have a filled/dense core. This is an out-of-focus artifact caused by it lying above the focal plane of the image because it is stacked on another fiber. (See Figure SI-1, Supporting Information, for further illustrations of this effect).

A 27 wt % sodium silicate solution was diluted 50-fold in distilled water and passed through a cationic exchange column (DOWEX 50WX4-50, Sigma-Aldrich), charged by flushing with hot distilled water, followed by 2 M HCl (aq) and cold distilled water. The resultant solutions were at pH 3-4, and were raised to a pH of 7.5 by addition of small amounts of the stock silicate solution. Fresh silicic acid was prepared in this way for each experiment. Template Silicification. Silica-coated peptide fibers were prepared in plastic containers at 20 °C by addition of 2 µL Si(OH)4 solution to 20 µL of buffered aqueous SAF solutions, to give final concentrations of Si(OH)4 ≈ 2 mM. The samples were slowly inverted (at 4 rpm) to ensure that the mixture remained homogeneous at all times. Typically, the mixtures were left overnight at 20 °C to produce well-defined silicified fibers. As described in the text, in some experiments the peptide template was removed by proteolysis. For this, 1 µL of a 100 µM chymotrypsin solution (containing 1 mM HCl, 2 mM CaCl2 in ultrapure water) was added to 100 µL of a dispersion of the silicified peptide fibers, and the mixture incubated at 20 °C for different lengths of time. Stability Studies. The effects of temperature, ionic strength and pH on standard and silicified SAFs were investigated by exposing dispersions of the samples to heat (20-60 °C) for 30 min; by addition of KCl to final salt concentrations of 50-800 mM for 1 h at 20 °C; and by incubation at 20 °C for 1 h in MOPS adjusted with HCl and NaOH to pH values away from neutral, but in the range 6-9. The extent of fiber loss and damage was judged qualitatively by TEM. In addition, the proteolysis reactions were monitored by CD spectroscopy as a function of time. Preparation of Polymer-Coated Silicified SAFs. Dispersions of silicified peptide fibers were added to a solution of the cationic polyelectrolyte, PDADMAC (1 wt % in distilled water), to give SAF/polymer volumetric ratios of 1:1 or 1:5. The resulting structures were removed for TEM analysis after 20 min. Circular Dichroism (CD) Spectroscopy. CD measurements were made using a JASCO J-810 spectropolarimeter fitted with a Peltier temperature controller. Spectra and data as a function of temperature were recorded as described previously.46 Typical parameters used

were: spectral range ) 190-260 nm; bandwidth ) 1 nm; response time ) 2 s; ramping rate ) 1 °C/min; and a cuvette with a 1 cm path length used throughout. Transmission Electron Microscopy (TEM). Droplets (8 µL) were applied to carbon-coated copper specimen grids (Agar Scientific Ltd., Stansted, UK) for 30 s, and then dried from underneath using filter paper. Negative-stain TEM samples were treated with filtered aqueous uranyl acetate (1%). Grids were examined using a JEOL JSM 1200EX TEM operating at an accelerating voltage of 120 keV. Digital images were acquired with a charge-coupled device camera (Soft Imaging System Megaview II), dimensional analysis carried out using the JEOL extended TEM program and energy dispersive X-ray (EDX) analysis using Oxford Instruments X-ray Analysis ISIS300).

Results and Discussion Standard SAF Preparation and Silicification. SAFs were produced using standard conditions: peptides SAF-p1 and SAFp2a, each at 100 µM, were incubated together overnight at 20 °C in aqueous buffer at pH 7.4. TEM images of uranyl acetatestained samples showed fibers that were uniform in width, straight, and generally intact after air-drying onto the grid, Figure 1A; morphological deviations such as bends, kinks, branches and breaks were generally not observed. In addition, a previously reported surface striation pattern of 4.3 nm52 was observed on the fibers. All of these features indicated the SAFs assembled faithfully as described elsewhere.46,52 Addition of silicic acid to final concentrations of 2 mM to dispersed, preformed SAFs produced visible increases in turbidity within 5 min, suggesting that silica polymerization was promoted in the presence of the peptide fibers. Unstained TEM, Figure 1B, (52) Papapostolou, D.; Smith, A. M.; Atkins, E. D. T.; Oliver, S. J.; Ryadnov, M. G.; Serpell, L. C.; Woolfson, D. N. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10853–10858.

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Figure 2. Deposition of silica on the SAF templates. Plots showing the dependence of fiber thickness with (A) Si(OH)4 concentration and (B) reaction time at [Si(OH)4] ) 2 mM. Lines added to guide the eye.

and EDX analysis (Figure SI-2, Supporting Information) of the resulting samples indicated that silica deposition was associated specifically with the surface of the peptide fibers to produce electron-dense composites consisting of a peptide core enclosed within a continuous silica shell. Under standard conditions (100 µM SAF peptides, 18 h, 20 °C, pH 7.2-7.4), the matured peptide fibers had an average diameter of 82 nm (n ) 307). After silicification (2 mM Si(OH)4, 18 h, 20 °C), this increased to 252 nm (n ) 528). The silicified SAFs were of the same length as the peptide templates and existed as discrete rods with smooth surfaces. Negligible levels of nontemplated silica were obtained under these conditions. The thickness of the silica coating deposited on the SAFs could be controlled by changes in silicic acid concentration, reaction time, or both. The layer thickness increased to produce fibers of 165 to 305 nm in diameter as the Si(OH)4 concentration was raised from 0.5 to 4 mM, after which the thickness of the inorganic coating remained unchanged, or decreased marginally (Figure 2A). This indicated that nucleation of silica specifically on the cationic peptide template was competitive over bulk precipitation at relatively low supersaturation levels, while concentrations of Si(OH)4 above 4 mM increased the level of nontemplated deposition associated with hydrolysis/condensation reactions occurring in solution. Consistent with this, electron micrographs of samples prepared at higher concentrations of Si(OH)4 showed higher backgrounds of amorphous silica precipitate. Increased thickness of the hybrid structures were also observed for samples removed at times up to ∼100 min during the reaction with 2 mM Si(OH)4, Figure 2B. Increased reaction times did not significantly change the thickness of the silica coating. Stability and Removal of the SAF Template. Circular dichroism (CD) spectroscopy provides a convenient measure of peptide secondary structure in peptides and proteins. The standard SAFs are characterized by distorted and attenuated CD spectra due to light scattering associated with chiral objects with dimensions comparable to the wavelength of light being used. (Figure 3A)46 Nonetheless, previous studies have shown by a combination of peptide redesign and biophysical studiessnotably X-ray diffractionsthat the assembled SAFs are fully R-helical.46,52 Significantly, the assembled and silicified SAFs described herein showed similar distorted CD spectra, though silicified

SAFs gave greater attenuation, presumably because the increased thickness of the mineralized structures scattered more light, Figure 3A. In addition, temperature-dependent CD spectra showed that the midpoint of unfolding for the standard SAFs was 49 °C;47 and a similar value was determined for the silicified SAFs. The results were therefore consistent with the presence of the SAF template within the silicified fibers, and suggested that the peptides largely retain their R-helical conformation within the inorganic sheath. TEM images indicated that the peptide assemblies remained intact when the silica-coated fibers were exposed to various conditions known to disassemble unmodified SAFs. For example, the silicified fibers were stable at pH 8.5, compared with the standard SAFs that start to unfold outside pH range 7-7.5.47 Similarly, the peptide cores of the coated fibers tolerated higher salt concentrationssthey were visible by TEM up to 400 mMsthan the uncoated SAFs, which were stable only up to 50 mM salt. CD spectra were also used to study the disassembly of the template when exposed to the protease chymotrypsin, which cleaves polypeptides after tyrosine residues present in the SAFs. The CD signal from the nonsilicified fibers was lost approximately twice as quickly as that from the coated SAFs, Figure 3B; coated fibers had a half-life of approximately 24 min in the presence of chymotrypsin, compared to