pH-Controlled Hierarchical Self-Assembly of Peptide Amphiphile

Apr 6, 2015 - This pH-responsive system is able to self-assemble into micelles, nanofibers, and bundles nanofibers, providing a new perspective toward...
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pH-Controlled Hierarchical Self-Assembly of Peptide Amphiphile Yiren Chen,† Hui Xian Gan,‡ and Yen Wah Tong*,‡ †

NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456 ‡ Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576 S Supporting Information *

ABSTRACT: The self-assembling behavior of peptide amphiphiles can be leveraged upon to form complex nanostructures that have a wide range of applications. Herein we present a study on the lateral assembly of peptide amphiphile nanofibers into higher order structures by designing complementary attracting designer sequences, made up of oppositely charged amino acids, on the surface region of the nanofibers. The surface charge of the individual amino acids can be selectively altered as the pH is raised above or below their pKa value, and this enables the regulation of interfiber and intrafiber interactions. We demonstrated that pH which triggers alternating positively and negatively charged surface sequence enables interdigitating and bundling of nanofibers, while pH which results in negatively charged surface sequence discourages lateral assembly of nanofibers. This pH-responsive system is able to self-assemble into micelles, nanofibers, and bundles nanofibers, providing a new perspective toward the fabrication of novel nanostructures.

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PA in response to the change in condition of the medium. Zhang et al. have presented several work based on ionic complementary peptides that display alternating of ionic charge groups that can self-assemble into macroscopic matrices in the presence of salt.26−29 Guo et al. investigated a PA based on silk fibroin that self-assembles into well-dispersed nanofibers. It was found that at pH 8 the fibers were able to aggregate in a parallel fashion with each other to form bundles. The aggregation was thought to be driven by hydrogen bonding between surface phenolic hydroxyl groups.30 Similarly, Cui et al. have demonstrated the assembly of PA into twisted nanoribbons at low concentration. These nanoribbons laterally assemble via a combination of the hydrophobic collapse of the alkyl tails and hydrogen bonding between glutamate side chains at low pH into giant nanobelts.10 In another study, Goldberger et al. found that PA, displaying a hydrophobic epitope on the surface of self-assembled nanofibers, tends to interlock with neighboring fibers to form thicker fiber bundles.31 The suppression of such bundling could be achieved by electrostatic repulsion between charged sequences in the PA. On the other hand, Ghosh et al. have prepared PA that transforms from spherical micelles to nanofibers under pH reduction, stimulating the acidic environment in the presence of malignant tissue.32 The peptide sequence was carefully designed such that micelles to nanofibers transition can be controlled based on the balance between attractive forces between β-sheet-forming and hydrophobic regions and repulsive forces of deprotonated glutamic

he hierarchical self-assembly of collagen molecules into triple helices, fibrils, and fiber bundles is an inspiration for the preparation of higher ordered nanostructures using biomolecules. The self-assembly process represents a spontaneous organization of supramolecular building blocks in a bottom-up nanofabrication of multidimensional and multiscale structures.1−3 Complex self-assembly processes with hierarchy levels have been realized in different systems such as stackable nanotoroids that form tubular and superhelical nanostructures4 and titanate nanotubes laced with amylose to form nanofibers that align to assemble laterally and longitudinally into hexagonal crystal domains.5 Diphenylalanine, a simplest smallmolecular weight building block derived from Alzheimer’s βamyloid polypeptide, has shown capabilities of template-free assembly into hexagonal peptide microtubes by thermal annealing6 or under the influence of a dipolar electric field.7 Amphiphilic peptide building blocks have been designed to spontaneously aggregate to form a variety of supramolecular self-assembled nanostructures such as nanotapes,8−10 nanoribbons,9,11−13 and nanofibers.14−18 Peptide amphiphile (PA), an oligopeptide functionalized with alkyl chains, was shown to be able to self-assemble into high-aspect-ratio cylindrical nanofibers upon triggering via light,19 pH,20,21 ions,22 and enzymes.23,24 Such a self-assembly process was found to be driven by formation of parallel β-sheets along the axis of the nanofibers.25 This highlights the importance of various noncovalent interactions for a self-assembly process. The realization of hierarchical self-assembly therefore hinges upon rational control of different noncovalent interactions that promotes aggregation at different stages of nanostructure formation. Several studies observed hierarchical self-assembly of © XXXX American Chemical Society

Received: December 24, 2014 Revised: March 24, 2015

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lysine and arginine residues in the PA design allowed us to selectively screen the charges of lysine residues at a pH between the pKa values of the two amino acids, thus isolating intrafiber interactions that elongate the nanostructure and interfiber interactions that promote lateral association of aligned nanofibers. Furthermore, the arginine and aspartic acid residues are separated by an alanine residue to prevent the neutralization of the side groups by proximity and thus allowing the interdigitation of surface region sequences when nanofibers align themselves in a supramolecular lateral self-assembly. Alanine was chosen over the conventional glycine as the spacer residue to reduce the flexibility and helical propensity of the surface region, thereby promoting interdigitation.56 To investigate the role of such complementarily attracting sequence in lateral assembly, we designed a series of PA with varying length of the alternating arginine and aspartic acid sequence. PA-1, PA-2, and PA-3 have the same peptide amphiphilic backbone but differ in the number of RADA repeats in the surface region (Scheme 1). These PAs are expected to self-assemble hierarchically to micelles, nanofibers, and bundles. Upon dissolution of the PAs, we observed the micelle formation through dynamic light scattering (DLS) measurements, circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM). At pH near the pKa of aspartic acid, DLS measurements suggest the self-assembly of the PA into micelles in solution given their small hydrodynamic radius (Table S1). This corresponds to the small spherical structures observed in TEM (see Figure S1 in the Supporting Information). The CD spectra of these PAs displayed predominantly random coil signals (see Figure S2) and are indicative of the micelle formation according to previous studies.22 The amphiphilic nature of the PAs is likely the driving force of the self-assembly while the electrostatic repulsion between the lysine residues in the charged region prevented the formation of cylindrical nanofibers. We thus establish the ability of the first pH switch at around pH 4 to trigger the self-assembly of the PAs into micelle upon dissolution. The second pH control was designed to be triggered by raising the pH of the medium above the pKa of both lysine and arginine. Here, the charges on lysine side chains were neutralized and allowed formation of nanofibers. The elongation of the micelles into nanofibers was observable in the significant change in hydrodynamic radius of the nanostructures from DLS measurements (Table 1). The zaverage sizes at pH 13 were similar for all PAs and were representative of the hydrodynamic radius of the nanofibers formed. Although the DLS measurements inherently adopt a spherical model, the increase in hydrodynamic radius gave us an indication of the larger geometries of the nanostructures within the solution. All PAs exhibit characteristic β-sheet signals in their CD spectra with a positive peak around 195 nm and a negative peak around 215 nm (see Figure S2). The hydrogen bond arrangements arising from the formation of parallel βsheets along the axis of the nanofibers has been shown to be the driving force of the elongation of the micelles into nanofibers.25,57 More conclusively, well-dispersed nanofibers were observed from TEM images. The nanofibers were randomly distributed single nanofibers that do not show any aggregation or lateral assembly (Figure 1). Taken together, the second pH switch at pH 13 proved to be successful in initiating the selfassembly of PA micelles into PA nanofibers. The pKa values of the side groups of lysine and arginine are 10.67 and 12.1,

acids. Although studies have demonstrated bundling of PA nanofibers, there has not been any systematic investigation on PA designs that direct lateral assemblies of one-dimensional nanostructures into higher order constructs. A flexible selfassembly system would provide a new toolbox for the formation of more complex nanostructures with great potential in molecular electronics,33−38 biochemical sensors,39,40 drug delivery,41−47 and tissue engineering.48−53 Particularly, pHcontrolled self-assembly offers enhanced feasibility and convenience when existing pH is leverage upon as internal stimuli. Typically, PA consists of four segments: (i) a hydrophobic alkyl tail, (ii) a β-sheet forming region, (iii) a charged region, and (iv) a surface region (Scheme 1). It has been reported that Scheme 1. Chemical Structure of PA-1 and the Sequence of PA-1 to PA-6a

a

The design of PAs has a similar backbone that consists of three segments: a hydrophobic region of hexadecanoic acid, a β-sheet forming region of four consecutive alanine residues, and a charged region of four consecutive lysine residues. Only the surface region varies to study the role of complementary interactions in the lateral assembly of nanofibers.

a force balance between the hydrophobic collapse of the alkyl tail and the electrostatic repulsive charges arising from the charged region results in the self-assembly of PA molecules into nanomicelles. 21 In our PA design, we have selected hexadecanoic acid as the alkyl tail and a polyalanine sequence from spider fibroin in the β-sheet forming region.54 We also inserted four lysine residues in the charged region to ensure the self-assembly of PAs into micelles under acidic conditions. At higher pH, the screening of the repulsive forces on charged residues immediately adjacent to the β-sheet region is expected to allow the formation of parallel β-sheet that elongates the micelles into cylindrical nanofibers. Moreover, it was reported that a minimum of four charged residues in the charged region will minimize interdigitation due to unintended hydrophobic interaction between the sequences in the surface region.31 We hypothesize that a complementarily attracting designer sequence at the surface region would be able to promote lateral assembly of nanofibers into higher order structures. We represent this segment with an alternating arginine and aspartic acid sequence in the surface region of the PA. At pH below the pKa of arginine, this sequence should present an alternating pattern of positive and negative charges on the surface of the nanofiber. This might possibly provide attractive interactions between interdigitating PA molecules of neighboring nanofibers and promote the process of lateral assembly.55 The choice of B

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alternating positive and negative charge sequence at the surface region that is complementarily attractive to neighboring nanofibers and thus promotes their lateral assembly. The PA solutions at pH 11 appeared more opaque due to light scattering from larger aggregates formed (see Figure S3). To investigate the aggregation of the nanofibers, DLS measurements of the PAs were taken at pH 11. A spike in z-average sizes for all the PAs was recorded at pH 11, and this could indicate an aggregation of nanofibers within the solution (see Table S1). The particles measured were at least 2−10 times larger at pH 11 than at pH 13, and there was also an increase in the change in z-average from pH 13 to pH 11 from PA-1 to PA3 (see Figure S4). This could be due to the aggregation of the nanofibers into bundles and that the bundling effect increases with the lengthening of the alternating arginine and aspartic acid sequence. The extent of nanofibers bundling was observed to vary among TEM images of different samples (Figure 2).

Table 1. Zeta Potential Values of PAs at Various pH Obtained from DLS Measurements (Concentration of PAs Used Was 0.25 mM) PA

pH

zeta potential (mV)

zeta potential deviation (mV)

PA-1

4 11 13 4 11 13 4 11 13 4 11 13 4 11 13 4 11 13

12.10 0.08 −16.2 34.00 −0.16 −31.60 44.60 0.10 −44.40 33.1 −6.94 −31.20 27.70 −1.52 −37.20 29.1 −8.79 −34.50

0.781 0.968 0.681 0.503 0.506 0.907 3.54 0.221 0.551 0.700 0.447 1.62 1.18 1.37 1.25 3.72 1.29 0.56

PA-2

PA-3

PA-4

PA-5

PA-6

Figure 1. (a−c) TEM images of PA-1, PA-2, and PA-3 at pH 13. Nanofibers self-assembled from the PAs are well-dispersed and do not show any signs of aggregation. Scale bar = 200 nm. (d) Histogram of average bundle width of PA-1, PA-2, and PA-3 at pH 13 (n > 120).

Figure 2. (a−c) TEM images of PA-1, PA-2, and PA-3 at pH 11. Nanofibers appear to aggregate in parallel fashion into network of bundles. Inset of (b) is PA-2 at pH 11 with 150 mM MgSO4. Nanofibers appear to be well-dispersed without the bundling effect seen without the salt addition. Scale bar = 200 nm. (d−f) Histogram of average bundle width of PA-1, PA-2, and PA-3 at pH 11 (n > 120).

respectively. At pH 13, both the side groups of lysine and arginine are expected to be deprotonated, leaving aspartic acid as the only charged residue in the surface region. The negative zeta potential measurements of these PAs at pH 13 (Table 1) confirmed the presence of negatively charged residues on the nanofiber surfaces. Consequently, the repulsive forces between negatively charged aspartic acid residues on the surface of the nanofibers could discourage any lateral assembly between nanofibers. Similar observations was reported in literature, whereby electrostatic repulsion between deprotonated carboxylate groups on glutamate side chains resulted in the dissociation of giant nanobelt.31 In another study, deprotonated phenol groups on tyrosine side chains give rise to similar electrostatic repulsion that resulted in well-dispersed nanofibers rather than parallel aggregates.30 The third pH switch was designed around pH 11 where the aspartic acid and lysine residues are deprotonated while the arginine residues remain protonated. This should produce an

Correspondingly, the bundling of the nanofibers seemed to be increasing in extent from PA-1 to PA-3 and was evident in the greater bundle widths. The observation of the aggregation in the PAs and the larger bundles formed for longer complementarily attracting sequence would suggest that the designed electrostatic attraction between the surface regions could be important in the lateral assembly of nanofibers. To elucidate the mechanism behind the bundling of the nanofibers, multivalent ions were added to PA-2 at pH 11 to disrupt the electrostatic attractions between the complementarily attracting sequences. Well-dispersed nanofibers were observed in TEM images, and the lack of nanofibers bundling suggests that the screening of charges on arginine and aspartic acid could disrupt the lateral assembly of the nanofibers (Figure C

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retained despite having a differently arranged surface region in PA-5 compared to PA-2. In addition, we wished to determine if the bundling effect would take place if the spacer amino acids in the surface region were substituted. On the basis of the findings above, we further synthesized PA-6, a PA bearing the fibronectin-mimetic sequences of RGDS, and designed it to be similar to PA-2 with two repeats of RGDS at the surface region. This cellbinding sequence bears similarities to the RADA tandem repeats used in the surface region and thus could be expected to be manipulated with the same pH triggers. CD studies identified the presence of β-sheet signals for PA-6 at both pH 11 and pH 13 (Figure S2), and DLS measurements showed a similar size trend to PA-2 (Table 1). TEM images confirmed the self-assembly of PA-6 to well-dispersed nanofibers at pH 13 and a further lateral assembly into bundles at pH 11 (Figure 4). The substitution of the spacer residues at the surface region of PA-2 did not appear to have any disruptive effects on the nanofiber bundling of PA-6.

2b). To further test the mechanism, PA-4 was synthesized. PA4 has a scrambled surface region where the charged residues are rearranged from a complementary attractive (+ − + −) sequence at pH 11 to a complementary repulsive (+ − − +) sequence. This would discourage any lateral assembly of the peptide nanofibers despite the presence of positive and negative charges on the RAADDAAR sequence in the surface region. Indeed, we observed a drop in z-average size at pH 11 in DLS measurements compared to the previous PA. Also, unlike the other PAs, we recorded similar z-average sizes for PA-4 at pH 11 and pH 13. Well-dispersed nanofibers that showed little lateral assembly at both pH 11 and pH 13 were also seen in TEM images (Figure 3). Therefore, the absence of apparent

Figure 4. (a, b) TEM images of PA-6 at pH 11 and pH 13, respectively. Nanofibers show similar lateral assembly at pH 11 and repulsion into dispersed single fibers at pH 13 as PA-2. Scale bar = 200 nm. (c) FESEM image of critically dried PA-6 hydrogel. Nanofibers are assemble laterally into a network of bundles with large pores. Scale bar = 1 μm. (d) Cryo-TEM image of PA-6 at pH 11. Bundling effect of nanofibers due to complementary interactions on the surface of nanofiber observed. Scale bar = 200 nm.

Figure 3. (a, b) TEM images of PA-4 at pH 11 and pH 13, respectively. Nanofibers appear to be well-dispersed without the bundling effect seen in PA-2. Scale bar = 200 nm. (c) Histogram of average bundle width of PA-4 at pH 11 (n > 120). (e, f) TEM images of PA-5 at pH 11 and pH 13, respectively. Nanofibers show similar bundling effect as PA-2 at pH 11. Scale bar = 200 nm. (f) Histogram of average bundle width of PA-5 at pH 11 (n > 120).

In the above experiments, the aggregation of PAs at pH 11 appeared to be in a network of bundles rather than a general collapse into a large globular structure. This is a characteristic that hints of strong interaction between stiff nanofibers.55 The strong interaction would likely be provided by the electrostatic attraction between the designed sequences in the surface region of the PA. Neighboring nanofibers aggregate in a parallel fashion as the PA molecules interdigitate and stabilize the bundles through the said attractive forces. These interactions do not exist when the alternating charges are changed from attractive interaction to repulsive interaction between interdigitating PA molecules. This was proven by the absence of nanofiber bundles when the positive charges in the surface region were removed by the deprotonation of arginine residues for all PAs at pH 13. A similar observation was found when the complementary alternating-charge sequence was rearranged in PA-4 to give repulsive interactions between aggregating nanofibers (Figure 5). We therefore postulate that the bundling

nanofiber bundling at pH 11 upon the addition of multivalent ions and the scrambling of the sequence in the surface region of PA-2 validate the importance of the alternating arginine and aspartic acid sequence in promoting lateral assembly of nanofibers. We further designed PA-5 to investigate the necessity of the alternating design on the surface region. The surface region for PA-2 was rearranged in PA-5 to RARADADA to retain complementary attractiveness with neighboring nanofibers (+ + − −) while shifting away from the alternating (+ − + −) design. The characterization of PA-5 yielded results similar to PA-2 at pH 11 and 13. DLS measurements showed a larger zaverage at pH 11 than pH 13 (Table 1), and bundling of the nanofibers was also only observed at pH 11 under TEM imaging (Figure 3). These results suggest that lateral assembly occurs when the complementary attractive interactions are D

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MATERIALS AND METHODS

Materials. All peptides were purchased from Biopeptek Inc. (Malvern, PA) with a purity of >95%. The peptides were synthesized using a standard Fmoc solid phase peptide synthesis according to the literature.59 All chemicals and solvents were purchased from SigmaAlrich (St. Louis, MO) unless otherwise mentioned. Peptide Amphiphile Self-Assembly. The PA solutions were prepared by first dissolving the lyophilized PAs in small amounts of HFIP and dried over filtered nitrogen gas until a thin layer of PA is formed. Ultrapure water (18.2 MΩ, Milli-Q A10) was added, and the pH of the solution adjusted to 4 with HCl. The PA solution was sonicated for 1 h at a temperature of 70 °C to ensure complete dissolution of the PA. The self-assembly of the PA was then triggered by the addition of NaOH in a dropwise fashion with the pH of the solution closely monitored with a small-volume probe pH meter (Fisher Scientific FSAB15A). Circular Dichorism (CD). Circular dichorism measurements were carried out using a JASCO J-810 CD instrument to determine the secondary structures characteristic of the micelle and nanofibers formed from self-assembly of the peptide amphiphiles. Wavelength scans were taken of the PAs in water at the different pH at 0.5 nm intervals between 250 and 185 nm. The data were acquired at room temperature and averaged over at least five acquisition cycles. All the PAs were measured at a concentration of 0.25 mM with a 0.1 cm path length quartz cuvette. All samples were allowed to equilibrate for 8 h prior to the measurements to ensure similar aging effect across the PAs. The spectra for all samples were corrected by subtracting the baseline, and the data were expressed as the mean residue of ellipticity given in the units of degrees per cm2 dmol−1. Transmission Electron Microscopy (TEM). Freshly prepared PA solutions of concentration 0.75 mM at different pH were used for all TEM sample preparation. 6 μL of each solution was dropped unto a carbon-coated FORMVAR copper grid (200 mesh) for 10 min, and excess fluid was removed using filter paper to produce a thin film of sample. The samples were stained with 1 wt % phosphotungstic acid for 1 min before they were air-dried for 3 h. The grids were stored at 35% humidity level until observation using a TECNAI T12 electron microscope with 120 kV operating voltage. Cryo-Transmission Electron Microscopy (Cryo-TEM). 0.75 mM PA solutions prepared freshly at the required pH were used to prepare samples for vitreous ice cryo-TEM. 3 μL of each solution was pipetted onto a plasma-cleaned quantifoil TEM grid (Electron Microscopy Sciences) before being blotted for 1 s using a FEI Vitrobot Mark IV. The grid was then plunged into liquid ethane and transferred into liquid nitrogen for viewing under a TECNAI T12 with a Gatan cryo-holder. The T12 instrument was operating at 120 kV under the low dose mode for cryo-observation. Field Emission Scanning Electron Microscopy (FESEM). 6 μL of freshly prepared PA solutions of concentration 7.5 mM at pH 4 were dropped onto cleaved mica surfaces stuck to SEM stubs via carbon tapes. The stubs were transferred to a sealed container with troughs of aqueous ammonia for 30 min to induce the raise in pH via gaseous ammonia. The hydrogel formed through the gas induction was dried at critical point using a Tousimis Autosamdri-815 instrument and coated with platinum before observing it under a JEOL JSM-740 instrument. Dynamic Light Scattering (DLS) and Zeta Potential. The zeta potentials and average z diameters of the PA aggregates were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern UK) with suitable viscosity and refractive index at 25 °C. Size measurements were obtained using small volume disposable micro cuvettes (ZEN 0040), and zeta potentials measurements were obtained using disposable folded capillary cells (DTS 1061). The values reported correspond to the mean of z-average and zeta potential values taken from replicates.

Figure 5. Schematic diagram of lateral assembly of self-assembled nanofibers due to interactions between the surface region of interdigitating PA molecules from neighboring nanofibers. Charged residues arranged in a complementary-attractive fashion result in the bundling of nanofibers, and those in a complementary-repulsive fashion result in well-dispersed nanofibers.

of the nanofibers is due to attractive electrostatic interactions between complementary sequences designed in the surface region of the PA molecules. Besides complementary sequences at the surface region, other factors such as aging, high PA concentration, and drying effects during TEM sample preparation could contribute to the unspecific aggregation of the nanofibers. However, the PAs were dissolved to the same concentration of 0.75 mM and aged for 8 h before staining for TEM observation. Aggregation due to drying effects from TEM preparation should affect all the three PAs similarly and thus could not adequately explain the bundling of the nanofibers at pH 11 rather than pH 13. Moreover, a higher concentration of PA-6 solution (7.5 mM) formed a hydrogel at pH 11 as the self-assembled nanofibers entangled into a network. The PA-6 gel was dried at critical point and visualized under field emission scanning electron microscope (FESEM) to reveal the network of fiber bundles that are consistent with prior observations (Figure 4c). Nevertheless, cryo-TEM was performed to confirm the formation of fiber bundles in vitrified samples at pH 11. Samples for cryo-TEM were prepared by rapidly plunge freezing the specimen into liquid ethane and liquid nitrogen to eliminate any artifacts due to drying or staining agents used in TEM, allowing the visualization of any bundling effect in the native structures of the PAs.58 The nanofibers were observed to align themselves in parallel aggregates to form fiber bundles that resemble those observed under conventional TEM (Figure 4d). In summary, we have designed a pH-responsive PA system that is capable of hierarchical self-assembly into micelles, nanofibers, and bundled nanofibers. The lateral assembly of nanofibers can be controlled by clever designs of complementary electrostatic attraction using oppositely charged amino acids pairing such as arginine and aspartic acid. Higher orders of hierarchical assembly with greater precision could result from further studies on higher specificity in the sequence design in the surface region. The controllable multiscale assembly presented here provides a new perspective toward the fabrication of novel nanostructures across different length scales. E

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(15) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004, 303, 1352−5. (16) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 2001, 294, 1684−8. (17) Caplan, M. R.; Schwartzfarb, E. M.; Zhang, S.; Kamm, R. D.; Lauffenburger, D. A. Control of self-assembling oligopeptide matrix formation through systematic variation of amino acid sequence. Biomaterials 2002, 23, 219−27. (18) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide. J. Am. Chem. Soc. 2002, 124, 15030−7. (19) Lee, H. K.; Soukasene, S.; Jiang, H.; Zhang, S.; Feng, W.; Stupp, S. I. Light-induced self-assembly of nanofibers inside liposomes. Soft Matter 2008, 4, 962−964. (20) Greenfield, M. A.; Hoffman, J. R.; de la Cruz, M. O.; Stupp, S. I. Tunable mechanics of peptide nanofiber gels. Langmuir 2010, 26, 3641−7. (21) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Peptide-amphiphile nanofibers: a versatile scaffold for the preparation of self-assembling materials. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5133−8. (22) Stendahl, J. C.; Rao, M. S.; Guler, M. O.; Stupp, S. I. Intermolecular forces in the self-assembly of peptide amphiphile nanofibers. Adv. Funct. Mater. 2006, 16, 499−508. (23) Dehsorkhi, A.; Hamley, I. W.; Seitsonen, J.; Ruokolainen, J. Tuning self-assembled nanostructures through enzymatic degradation of a peptide amphiphile. Langmuir 2013, 29, 6665−72. (24) Webber, M. J.; Newcomb, C. J.; Bitton, R.; Stupp, S. I. Switching of self-assembly in a peptide nanostructure with a specific enzyme. Soft Matter 2011, 7, 9665−9672. (25) Pashuck, E. T.; Cui, H.; Stupp, S. I. Tuning supramolecular rigidity of peptide fibers through molecular structure. J. Am. Chem. Soc. 2010, 132, 6041−6. (26) Zhang, S.; Rich, A. Direct conversion of an oligopeptide from a beta-sheet to an alpha-helix: a model for amyloid formation. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 23−8. (27) Yokoi, H.; Kinoshita, T.; Zhang, S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 8414−8419. (28) Ye, Z.; Zhang, H.; Luo, H.; Wang, S.; Zhou, Q.; Du, X.; Tang, C.; Chen, L.; Liu, J.; Shi, Y. K. Temperature and pH effects on biophysical and morphological properties of self-assembling peptide RADA16-I. J. Pept. Sci. 2008, 14, 152−162. (29) Zhang, S. Emerging biological materials through molecular selfassembly. Biotechnol. Adv. 2002, 20, 321−339. (30) Guo, H.; Zhang, J.; Xu, T.; Zhang, Z.; Yao, J.; Shao, Z. The robust hydrogel hierarchically assembled from a pH sensitive peptide amphiphile based on silk fibroin. Biomacromolecules 2013, 14, 2733−8. (31) Goldberger, J. E.; Berns, E. J.; Bitton, R.; Newcomb, C. J.; Stupp, S. I. Electrostatic control of bioactivity. Angew. Chem., Int. Ed. 2011, 50, 6292−5. (32) Ghosh, A.; Haverick, M.; Stump, K.; Yang, X.; Tweedle, M. F.; Goldberger, J. E. Fine-tuning the pH trigger of self-assembly. J. Am. Chem. Soc. 2012, 134, 3647−3650. (33) Liu, H.; Xu, J.; Li, Y. Aggregate nanostructures of organic molecular materials. Acc. Chem. Res. 2010, 43, 1496−508. (34) Djalali, R.; Chen, Y. F.; Matsui, H. Au nanowire fabrication from sequenced histidine-rich peptide. J. Am. Chem. Soc. 2002, 124, 13660− 1. (35) Tevis, I. D.; Tsai, W. W.; Palmer, L. C.; Aytun, T.; Stupp, S. I. Grooved nanowires from self-assembling hairpin molecules for solar cells. ACS Nano 2012, 6, 2032−40. (36) Faramarzi, V.; Niess, F.; Moulin, E.; Maaloum, M.; Dayen, J. F.; Beaufrand, J. B.; Zanettini, S.; Doudin, B.; Giuseppone, N. Lighttriggered self-construction of supramolecular organic nanowires as metallic interconnects. Nat. Chem. 2012, 4, 485−90.

ASSOCIATED CONTENT

S Supporting Information *

TEM images of PA-2, PA-4, and PA-5, circular dichroism spectra, photographs of glass vials containing PA solutions at different pH, and pH titration graph of z-average and zeta potential values. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], Fax (+65) 6779 1936 (Y.W.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support by the National University of Singapore (NUS) through grant R279000353112. Yiren Chen thanks the National University of Singapore Graduate School for Integrative Sciences and Engineering for his scholarship.

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REFERENCES

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DOI: 10.1021/ma502572w Macromolecules XXXX, XXX, XXX−XXX