Peptide Amphiphile Nanofibers Template and Catalyze Silica

Mar 21, 2007 - Natural biomineralization creates the most intricately stunning inorganic structures, such as those found in mollusk shells,1,2 sea urc...
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Langmuir 2007, 23, 5033-5038

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Peptide Amphiphile Nanofibers Template and Catalyze Silica Nanotube Formation Virany M. Yuwono and Jeffrey D. Hartgerink* Departments of Chemistry and Bioengineering, Rice UniVersity, 6100 Main St. MS. 60, Houston, Texas 77005 ReceiVed October 10, 2006. In Final Form: February 8, 2007 Hollow silica nanotubes with tunable dimensions have been synthesized by condensation of tetraethoxysilane (TEOS) on peptide-amphiphile nanofiber templates followed by calcination. Peptide-amphiphile nanofibers direct silica mineralization by providing nucleation sites and catalyze silica polymerization at their surface. The catalytic activities of peptide-amphiphiles containing lysine, histidine, or glutamic acid were compared and only peptide amphiphiles containing lysine or histidine were found to be good catalytic templates. Depending on the reaction conditions, and the size of the PA assembler, the nanotube wall thickness could be varied between 5 and 9 nm.

Introduction Natural biomineralization creates the most intricately stunning inorganic structures, such as those found in mollusk shells,1,2 sea urchin spine,3,4 cocolith,5 bone, teeth,6 and diatom cell walls.7 Such a high degree of organization is achieved, in part, through the presence of organic macromolecules, which control many aspects of the mineralization process, including crystal nucleation and growth.8 The success of nature in creating well-defined and precisely controlled inorganic structures is a source of inspiration for creating synthetic counterparts, especially in the area of nanotechnology. Biomimetic approaches typically involve the presence of organic molecules controlling the growth of inorganic materials.9-14 However, manipulation and organization of inorganic materials in vitro at the nanoscale remains challenging. Understanding the detailed function of biological macromolecules in conjunction with their design is pivotal in determining the appropriate organic systems needed to experimentally achieve the desired outcomes. Silica is an oxide which is prevalent in nature (e.g., diatoms) and technologically important. The synthesis of silica nanostructures has gained attention due to their potential applications in catalysis15,16 and electronics.17 The sol-gel chemistry of silica, in which tetraalkoxysilicate precursors are hydrolyzed and * To whom the correspondence should be addressed. Phone: (713) 3484142. E-mail: [email protected]. (1) Fu, G.; Qiu, S. R.; Orme, C. A.; Morse, D. E. De Yoreo, J. J. AdV. Mater. 2005, 17, 2678-2683. (2) Fu, G.; Valiyaveettil, S.; Wopenka, B.; Morse, D. E. Biomacromolecules 2005, 6, 1289-1298. (3) Berman, A.; Addadi, L.; Weiner, S. Nature 1988, 331, 546-8. (4) Berman, A.; Addadi, L.; Kvick, A.; Leiserowitz, L.; Nelson, M.; Weiner, S. Science 1990, 250, 664-7. (5) Black, M. Proc. Linn. Soc. London 1963, 174, 41-46. (6) Daculsi, G.; Menanteau, J.; Kerebel, L. M.; Mitre, D. Calc. Tiss. Int. 1984, 36, 550-5. (7) Coombs, J.; Volcani, B. E. Planta 1968, 82 280-292. (8) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: Oxford, U.K., 1989. (9) Yang, L.; Shen, Q.; Zhou, J.; Jiang, K. Mater. Chem. Phys. 2006, 98, 125-130. (10) Zhang, Z.; Gao, D.; Zhao, H.; Xie, C.; Guan, G.; Wang, D.; Yu, S.-H. J. Phys. Chem. B 2006, 110, 8613-8618. (11) Zhan, J.; Tseng, Y.-H.; Chan, J. C. C.; Mou, C.-Y. AdV. Funct. Mater. 2005, 15, 2005-2010. (12) Tanev, P. T.; Pinnavaia, T. J. Science 1996, 271, 1267-9. (13) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2002, 1, 169-72. (14) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature 2000, 403, 289-92.

condensed on organic templates has been extensively studied. Nanostructures of silica have been synthesized by this strategy utilizing various templates, including phospholipids,18 peptidic lipid,19,20 laurylamine hydrochloride,21 collagen fibers,22 organogels,23 and peptide fibrils.24 Peptide amphiphiles (PAs) are an attractive class of selfassembling systems for biomimetic mineralization. Due to their intermolecular hydrogen bonding and amphiphilic nature, PAs organize into long and stable cylindrical nanofibers in aqueous media, exposing a surface rich with functional groups.25-27 The self-assembly behavior of the PAs is governed in large part by the pH in relation to the pKa values of the amino acid residues included in the peptide design. PAs whose net repulsive charge overcomes the attractive interactions of hydrophobic packing and hydrogen bonding will not self-assemble. Elimination of or reduction of charges can be achieved by adjusting the pH or by addition of multivalent ions, for example Ca2+.28 The surface chemistry of the nanofibers is determined by the amino acids incorporated in the peptide sequence and can tolerate a wide range of natural and unnatural amino acids and their derivatives. Previously, PAs have been shown to promote nucleation and growth of hydroxyapatite27 and CdS.29 In this paper, we describe the templating effect of PA nanofibers in tetraethoxysilane (TEOS) polymerization as a versatile approach to synthesizing hollow (15) Kageyama, K.; Tamazawa, J.-I.; Aida, T. Science 1999, 285, 21132115. (16) Nagaraju, N.; Fonseca, A.; Konya, Z.; Nagy, J. B. J. Mol. Catal. A 2002, 181, 57-62. (17) Pender, M. J.; Sowards, L. A.; Hartgerink, J. D.; Stone, M. O.; Naik, R. R. Nano Lett. 2006, 6, 40-44. (18) Baral, S.; Schoen, P. Chem. Mater. 1993, 5, 145-147. (19) Ji, Q.; Iwaura, R.; Shimizu, T. Chem. Lett. 2004, 33, 504-505. (20) Ji, Q.; Iwaura, R.; Kogiso, M.; Jung, J. H.; Yoshida, K.; Shimizu, T. Chem. Mater. 2004, 16, 250-254. (21) Adachi, M.; Harada, T.; Harada, M. Langmuir 1999, 15, 7097-7100. (22) Ono, Y.; Kanekiyo, Y.; Inoue, K.; Hojo, J.; Nango, M.; Shinkai, S. Chem. Lett. 1999, 475-476. (23) Ono, Y.; Nakashima, K.; Sano, M.; Hojo, J.; Shinkai, S. Chem. Lett. 1999, 1119-1120. (24) 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. (25) Paramonov, S. E.; Jun, H.-W.; Hartgerink, J. D. J. Am. Chem. Soc. 2006, 128, 7291-7298. (26) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Proc. Natl. Acad. Sci. 2002, 99, 5133-8. (27) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294, 16841688. (28) Jun, H.-W.; Yuwono, V.; Paramonov, S. E.; Hartgerink, J. D. AdV. Mater. 2005, 17, 2612-2617. (29) Sone, E. D.; Stupp, S. I. J. Am. Chem. Soc. 2004, 126, 12756-12757.

10.1021/la0629835 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/21/2007

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Yuwono and Hartgerink

Table 1. Peptide Sequences PA

N-terminus

sequence

C-terminus

1 2 3 4 5

CH3(CH2)14COCH3(CH2)14COCH3(CH2)14COCH3(CH2)14COCH3(CH2)14CO-

A4K4 A4H4 A4E4 A8K4 A12K4

-CONH2 -CONH2 -CONH2 -CONH2 -CONH2

silica nanotubes. PAs are ideal templates since they allow us to observe the effects of single amino acid modifications while still maintaining nanofibrous morphology. PAs are easily synthesized using standard solid-phase Fmoc chemistry followed by acylation of the N-termini and offer potentially endless combinations of amino acid residues to achieve different interactions with TEOS or other silica precursors. Molecular Design. Two series of PAs were prepared (see Table 1). The first series was designed to test the effect of three different chemistries provided by the peripheral amino acids lysine, histidine, and glutamic acid, which are exposed on the surface of the nanofiber. PA1 contains four alanine residues which are known to readily adopt the necessary extended β-sheet conformation for PA assembly. These are followed by four lysine residues. Lysine is especially attractive considering the abundance of this residue in sillafins30,31 and silicateins,32 naturally occurring proteins responsible in the formation of biosilica in diatoms and sponges, respectively. The N-terminus is coupled to palmitic acid to provide the hydrophobic driving force for self-assembly, whereas the C-terminus is amidated to limit charges on the PA to only those found on amino acid side chains. This PA is expected to be soluble at acidic and neutral pH and to begin self-assembly as the positive charges on the lysine side chains are neutralized at high pH. PA2 is the same as PA1 except that the lysine residues are replaced with histidine, allowing it to self-assemble at much lower pH (for example at a pH of 7) where the histidines are neutral. Lysine and histidine were also incorporated into our study because of the presence of amine and imidazole groups, which may act as surface anchored catalysts and may substitute for ammonium hydroxide - a typical catalyst in the sol-gel reaction. In contrast, amine functional groups are not present in PA3, which uses glutamic acid for its four peripheral residues. This PA is only expected to assemble below a pH of 5. However, it has been previously shown26,33 that PAs which incorporate glutamic acid can be assembled by addition of divalent cations such as Ca2+. In this study, we use Ca2+ to assemble the PA at neutral pH which can be adjusted to the same basic pH as PA1 and remain self-assembled. The second series of PAs was designed to determine the effect of PA sequence length on mineralization dimensions while keeping other parameters, such as surface chemistry, constant. PA1 uses four alanine residues for a PA eight amino acids in length; PA4 uses eight alanine residues for a PA 12 amino acids in length; and PA5 uses 12 alanines for a total length of 16 amino acids. Experimental Methods Synthesis and Purification of Peptide Amphiphiles. The list of peptide sequences can be found in Table 1. Peptide amphiphiles were synthesized by using minor modifications to methods previously described.26,28 The amino acids and resins were purchased from Novabiochem, and other chemicals were purchased from Sigma. (30) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129-1132. (31) Kroger, N.; Lorenz, S.; Brunner, E.; Sumper, M. Science 2002, 298, 584-586. (32) Shimzu, K. C., J.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. 1998, 95, 6234-6238. (33) Beniash, E.; Hartgerink, J. D.; Storrie, H.; Stendahl, J. C.; Stupp, S. I. Acta Biomater. 2005, 1, 387-397.

Figure 1. Negative stain TEM image of PA1 at 1 mM showing the characteristic nanofibrous structure.

Figure 2. CD spectra of 1 mM PA solutions after self-assembly. Peptides were synthesized from Rink-amide-MBHA resins to provide C-terminally amidated peptides. Before cleavage from the resin, the peptides were acylated with a mixture of 2 equiv of palmitic acid, 2 equiv of N-[(1H-benzotriazol-1-yl)(dimethylamino) methylene]N-methyl-methanaminimum hexafluorophosphate N-oxide (HBTU), and 6 equiv of diisopropylmethylamine (DIEA) in dimethylformamide (DMF) for 12 h at room temperature. After repeating the reaction once, the ninhydrin test was used to confirm the completion of the reaction. Peptide amphiphiles were cleaved and deprotected in a mixture of trifluoroacetic acid (TFA), triisopropylamine, anisole, and water in 27:1:1:1 volumetric ratio for 3.5 h at room temperature. The PA was precipitated in cold diethylether, and the precipitate was lyophilized for 2 days. Since PA solubility in water is pH dependent, purification was achieved by dissolving the PAs in water, followed by filtration and precipitation by adjusting the pH to induce self-assembly. The precipitate was collected, washed, redissolved in water, and filtered. The final product was lyophilized again for 2 days. The PAs were characterized by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry: PA1 expected mass [M+H]+ ) 1053, observed mass ) 1053 PA2 expected mass [M+H]+ ) 1088, observed mass ) 1088. PA3 expected mass [M+Na]+ ) 1079, observed mass )1079. PA4 expected mass [M]+ ) 1337, observed mass ) 1337. PA5 expected mass [M]+ ) 1621, observed mass ) 1621. TEOS Polymerization. The synthesis of silica on the surface of PA nanofibers was carried out under a large variety of concentrations and reagent ratios as detailed in Table 2. In a typical experiment,

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Table 2. Experimental Conditions in TEOS Mineralization Studies sample

PA (µmol)

TEOS (µmol)

[TEOS]/[PA]

NH4OH (µmol)

Ca2+ (µmol)

H2O (µL)

EtOH (µL)

pH ((0.2)

PA1-1 PA1-2 PA1-3 PA1-4 PA1-5 PA1-6 PA1-7 PA2-1 PA2-2 PA2-3 PA2-4 PA2-5 PA2-6 PA2-7 PA2-8 PA2-9 PA3-1 PA3-2 PA3-3 PA3-4 PA4-1 PA4-2 PA4-3 PA4-4 PA5-1 PA5-2 PA5-3 PA5-4

0.2 0.2 0.2 0.2 2 2 2 0.2 0.2 0.2 0.2 2 2 2 2 2 2 2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

4 10 20 40 20 20 40 4 10 20 40 20 40 100 200 400 40 20 20 20 4 10 20 40 4 10 20 40

20 50 100 200 10 10 20 20 50 100 200 10 20 50 100 200 20 10 100 100 20 50 100 200 20 50 100 200

0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 30 0 30 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0.2 0.2 0 0 0 0 0 0 0 0

200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200

200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200

10 10 10 10 10 10 10 7 7 7 7 7 7 7 7 7 10 10 10 10 10 10 10 10 10 10 10 10

lyophilized PA was dissolved in water and mixed with an equal volume of pure ethanol. Ammonium hydroxide (29.3%) was used as catalyst in some cases. When NH4OH was used as catalyst, the corresponding amount was added to the reaction mixture, otherwise, NaOH was used to adjust the pH to promote self-assembly, followed by addition of TEOS. CaCl2 was only used in the experiments involving PA3. The molar ratios between PA and TEOS ranged from 10 to 200. The reactions proceeded under continuous stirring for up to a week at room temperature, with periodic sample collection. The silica coated PA was centrifuged for 30 min at 13 000 rpm, washed twice with 2 mL absolute ethanol, and dispersed by sonication for 15 min in 600 µL of ethanol before storing. Aliquots from this solution were taken for calcination and TEM experiments. Calcination. Silica coated PA nanofibers in ethanol were placed in a metal cup at room temperature until ethanol evaporated. The resulting solid was placed in a high-temperature oven and heated from room temperature to 823 K at a rate of 5 K/min and the temperature was held constant at 823 K for 8 h. Afterwards, the samples were cooled to room temperature. TEM Sample Preparation. 10 µL of sample was dropped on the holey carbon grid, and the excess solution was removed after 1 min. Samples of PA without silica were negatively stained twice with 2% (w/v) phosphotungstic acid. The excess solution of the stain was removed after 1 min. Calcinated silica tubes were redispersed in ethanol and dropped on the TEM grid as described above. No stain was applied on the samples of silica coated PAs and calcinated tubes. Samples were analyzed under JEOL 2010 TEM at 200 kV.

Results Lyophilized powders of PA were dissolved in water to a concentration of 1 mM. Solutions of PA 1, 4, and 5 were then adjusted to pH 10 by addition of NaOH to induce self-assembly. PA2 was adjusted to pH 7 which was sufficient to trigger selfassembly due to the lower pKa of histidine as compared to lysine. PA3 was adjusted to pH 7 to dissolve and was subsequently self-assembled with the addition of CaCl2 at a 1:1 molar ratio with the PA. The resulting viscous suspension was adjusted to pH 10 with NaOH. Negatively stained TEM images of all of the PAs show the characteristic nanofibrous morphology of the PAs. PAs 1-3 were found to have diameters of approximately 6.5

Table 3. Silica Nanotube Dimensions at 100:1 [TEOS]:[PA] after 7 Days Incubation

samplea

inner diameter (nm)

coating thickness (nm)

outer diameter (nm)

PA1-3 2-3 3-3 4-3 5-3

4.7 ( 0.4 4.5 ( 0.4 none 4.2 ( 0.5 4.5 ( 0.6

7.6 ( 0.6 4.9 ( 0.4 none 8.9 ( 0.8 9.2 ( 0.9

19.9 ( 1.4 14.3 ( 1.2 none 22.0 ( 2.1 22.9 ( 2.4

a

See Table 2 for specific preparation conditions.

nm, whereas the longer peptide amphiphiles 4 and 5 were found to have diameters of 7.1 ( 0.4 and 7.2 ( 0.4 nm, respectively. Figure 1 shows typical nanofibers from PA1. The secondary structure of the PAs in aqueous solution was investigated by circular dichroism spectroscopy. Figure 2 summarizes the CD spectra for all PAs at self-assembly conditions at a concentration of 1 mM. The spectra display a negative peak between 217 and 223 nm and a maximum between 192 and 199 nm, consistent with the formation of β-sheet conformation in all cases as is typical for PAs. TEOS Mineralization Experiments. PA nanofibers, as prepared above, were used as templates for TEOS polymerization. It has been previously reported that the thickness of silica coating on a template is related to the reaction time and TEOS concentration.34 In our study, the effects of these factors in addition to a change in fiber surface chemistry and length of the peptide region were examined. Generally, a ratio of 100 TEOS molecules per PA molecule and a reaction time of one week gave the best results (uniformity of coating and reproducibility). The results are summarized in Table 3. In the absence of PAs, holding other parameters constant, we observed the formation of amorphous silica. The deposited silica did not change appreciably in shape or quantity between 1 day and 3 days of reactions. At no time were silica fibers of any type observed (Figure S1). (34) Yin, Y.; Lu, Y.; Sun, Y.; Xia, Y. Nano Lett. 2002, 2, 427-430.

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Figure 3. TEM images of silica coated PA1 samples after incubation in TEOS. [TOES]/[PA] ) 100, 7 days of incubation.

Figure 4. TEM images of silica coated PA2 samples after incubation in TEOS. [TEOS]/[PA] ) 100, 7 days of incubation.

Peptide Amphiphile 1: AAAAKKKK. After addition of TEOS, the PA suspension remained clear for 24 h. TEM analysis revealed fibers lightly coated in silica but not thick enough for distinct inner and outer diameters to be measured. After 7 days, silica-coated fibers were clearly visible, especially for PA to TEOS ratio of 1:50 and above (preparations PA1-2 through PA14). EDX of the sample indicated the presence of silica (Figure S5). The samples produced nanotubes with similar inner diameters just less than 5 nm. The coating thickness increased as the amount of TEOS in the mixture increased, as expected. Addition of NH4OH, a typical catalyst for TEOS polymerization, accelerated the rate of silica deposition on the nanofibers but did not significantly alter the final product formed (inner or outer diameter of silica deposition). Peptide Amphiphile 2: AAAAHHHH. TEOS hydrolysis and condensation on PA2 proceeds under mild conditions (pH ) 7), similar to conditions in biological systems. TEM analysis after 7 days of incubation revealed long fibers coated in silica (Figure 4). The most favorable conditions for PA2 were demonstrated by samples PA2-3. After a week, the resulting silica inner-core diameter was 4.5 ( 0.4 nm and the coating thicknesses was 4.9 ( 0.4 nm. As in the case of PA1, the thickness of the silica shell increased as the TEOS to PA ratio was increased. Peptide Amphiphile 3: AAAAEEEE. When PA3 nanofibers were incubated with TEOS, no silica templating was observed. Instead, the mixture exhibited nonspecific aggregation, which was imaged by TEM (Figure 5a). However, when NH4OH was

Yuwono and Hartgerink

added as a catalyst, silica coated fibers were formed although the coating was uneven and substantial silica was observed in regions without PA while other PA nanofibers were essentially uncoated. Without the addition of Ca2+, PA3 did not form nanofibers at pHs above the pKa of Glu, due to the presence of repulsive negative charges on glutamate ions. TEM analysis of disassembled PA3 incubated in TEOS shows amorphous silica and unstructured PA aggregates (Figure S2). The silica did not display any specific interaction preference with the PA aggregates, consistent with the results from self-assembled PA3. Peptide Amphiphile 4: AAAAAAAAKKKK. With the use of eight alanine residues in the peptide sequence, the fiber diameters were observed to increase. As expected, PA4 also catalyzes the reaction as PA1 does, indicated by the formation of silica coating on PA fibers. The progress of mineralization was followed for one week. Silica growth was fastest during the first 24 h, reaching a thickness of 7.2 ( 0.4 nm. After 7 days, the coating thickness grew to 8.9 ( 0.8 nm. As with the other PA templates, the core diameter became smaller as the reaction progressed. Peptide Amphiphile 5: AAAAAAAAAAAAKKKK. PA5 utilizes 12 alanine residues, expanding the fiber diameter further. All of the samples from PA5 formed silica nanotubes. The reaction progress was monitored for 7 days. As in the case of PA4, the coating thickness increased dramatically during the first day, reaching 7.6 ( 0.8 nm. During the next 3 days, the reaction slowed, gaining less than 1 nm in thickness. The reaction progressed to yield a final thickness of 9.2 ( 0.9 nm. As previously observed, the inner diameter became smaller as time passed yet reached a minimum of slightly less than 5 nm. Calcination. Each of the PAs which showed a good ability to template the polymerization of TEOS (PA1, 2, 4, and 5) were calcincated by heating to 823K for 8 h. Figure 8 shows hollow silica nanotubes after calcination from PA2. The silica morphology and dimensions remain largely intact after removal of the template; however, the thickness of the silica nanotubes increases slightly, consistent with a previous report.24

Discussion Five new PAs were prepared and all were found to selfassemble into uniform nanofibers as observed by TEM (Figure 1). All these fibers display the β-sheet secondary structure common to the PA nanofibers (Figure 2). PA2 and PA3 are of particular note because they self-assemble under unusual conditions. PA2 uses histidine, not previously used in peptideamphiphile studies, as its functional amino acid, and its pKa near 6 allows it to self-assemble at pH 7, a characteristic which may be useful in other studies in which self-assembly at physiological pH is desirable. PA3 uses glutamic acid as its functional amino acid and as such has two assembly mechanisms available to it: pH triggered assembly26 and multivalent cation assembly.26,33 After assembly triggered with calcium chloride, PA3 is able to retain its fibrous structure at a very broad range of pH, including 10, which is used in this study. After assembly, the PAs are treated with TEOS and periodically monitored for mineralization. PA1, 2, 4, and 5 displayed good templating of silica on the surface of their fibers and had little or no nonspecific TEOS polymerization. PA3 was found to have very little polymerization. The silica appeared to be distributed in a nonspecific fashion throughout the sample. This failure was expected since PA3 does not contain any amines to catalyze or template the condensation reaction. Addition of NH4OH to the PA3 solution allowed TEOS polymerization to proceed. However, the resulting morphology of silica deposition

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Figure 5. TEM images of silica coated PA3 samples after incubation in TEOS. [TEOS]/[PA] ) 100. (a). Without NH4OH after 7 days, (b) with NH4OH after 1 day.

Figure 6. TEM images of silica coated PA4 samples after incubation in TEOS. [TEOS]/[PA] ) 100, 1 day of incubation (left) 7 days of incubation (right).

Figure 7. TEM images of silica coated PA5 samples after incubation in TEOS. [TEOS]/[PA] ) 100, 4 days of incubation.

suggests a nonspecific adherence to the surface of the PA nanofiber and resulted in large amounts of nonspecific silica deposition. The diameters of PA nanofibers are proportional to the length of the PA molecule. However, as observed from Table 3, the inner diameter of silica tubes appears to be largely independent of the size of the PA. Although the inner diameter of the nanotubes becomes smaller as the incubation time increases, which is more pronounced for longer PAs (PA4 and PA5), all of the templating PAs show a minimum inner diameter of just less than 5 nm. This

Figure 8. Silica nanotubes after calcination of fibers templated from PA2.

value corresponds approximately with double the expected length of the PA aliphatic tail plus the four core alanine residues. As shown in Scheme 1b, the PA nanofiber has three distinct regions: the innermost hydrophobic core, a middle region composed of structured amino acids engaged in β-sheet-like hydrogen bonding25 and a peripheral region of amino acids without strong structural requirements. The innermost penetration of the silica into the PA appears to stop in the intermediate structured region of the PA. This effect is likely caused by the diffusion of TEOS precursor deep into the peptide region, which enables growth inward and outward from the nucleation sites at the surface of the nanofibers. The extent of this diffusion is also dependent on the concentration of TEOS in the reaction mixture. The thickness of the silica layer is more straightforward to analyze. It is proportional to the length of the PA and becomes larger as the incubation period is increased. In agreement with published work of Yin et al.,34 the reaction proceeds fastest during the early time frame, in our study 1 day, in which the thickness accumulation reaches up to 8.6 ( 0.9 nm for the longest PA. Catalytic Activity. At pH 7 used for the histidine containing PA2, and pH 10 for the lysine containing PA1, the PA nanofiber surface will be composed of a mixture of positively charged and neutral species since these pHs are near the pKa of their respective amino acids. TEOS will undergo hydrolysis catalyzed by the neutral primary amine of lysine or the indole nitrogen of histidine. This is consistent with previous reports.35,36 The resulting silicic (35) Zhou, Y.; Shimizu, K.; Cha, J. N.; Stucky, G. D.; Morse, D. E. Angew. Chem., Int. Ed. 1999, 38, 780-782.

5038 Langmuir, Vol. 23, No. 9, 2007 Scheme 1. Depiction of Synthetic Strategya

Yuwono and Hartgerink

PA3, which contains no amine, does not show any catalytic activity since it does not catalyze hydrolysis nor does it concentrate the polymerizing TEOS deriviative at its surface since it lacks positive charges. Even after 7 days of incubation, very little silica is observed and it is not specifically associated with PA nanofibers (Figure 5a) but is instead randomly distributed throughout the sample. Addition of an external catalyst such as NH4OH dramatically accelerates silica polymerization, but the silica is formed in a nonspecific fashion, some forming thick irregular coats on the PA nanofibers, whereas much of it is unassociated with the fiber (Figure 5b).

Conclusions

a (a) Chemical structure of PA1 before self-assembly. (b) Peptide amphiphiles are self-assembled into cylindrical nanofibers which expose specific chemical functionality. Shown is the hydrophobic core in gray, structured beta-sheet region in blue, and the conformationally flexible region in green. (c) This is used as a template for TEOS polymerization. (d) The organic portion can then be removed by calcination if desired.

acid or partially hydrolyzed TEOS will deprotonate to give a negatively charged species. This negative ion will then preferentially interact with the residual positive charge on the nanofiber, acting as the counterion to the positive charge on the fiber and enhancing its local concentration at the fiber surface. Polymerization to form silica then takes place nearly exclusively at the fiber surface resulting in a silica shell around the PA nanofiber. (36) 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.

We have demonstrated a versatile approach to synthesize silica nanotubes by sol-gel condensation of TEOS on the surface of PA nanofibers. The PA architecture allows us to modify the surface chemistry of the fiber. The modified surface chemistry allows the PAs to assemble under different conditions (pH 7 and 10) and also to polymerize the TEOS substrate under different conditions (pH 7 or 10) without the need for an auxiliary amine catalyst. Elimination of the amino and imidizole chemical functionality eliminated the templating effect of the PA nanofibers demonstrating that the fiber acts as more than a simple surface on which for silica to deposit, but also as a nucleating agent and catalyst. Moreover, the catalytic moiety is contained within the side chain of the respective lysine or histidine and not the backbone of the peptide. Modification of the length of the PA allows a controlled increase in the thickness of silica coating while the inner tube diameter reaches a minimum value equal in size to approximately twice the length of the aliphatic tail and four core structured alanine residues. Calcination of the templated silica allows the formation of nanostructured silica tubes. Acknowledgment. This work is supported by the Welch Foundation research Grant C-1557 and the Searle Scholars Program. Supporting Information Available: TEM and EDX spectra in Figures S1-S5. This material is available free of charge via the Internet at http://pubs.acs.org. LA0629835