Effects of Salt Concentrations of the Aqueous Peptide-Amphiphile

Sep 8, 2014 - Effects of Salt Concentrations of the Aqueous Peptide-Amphiphile Solutions on the Sol–Gel Transitions, the Gelation Speed, and the Gel...
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Effects of Salt Concentrations of the Aqueous Peptide-Amphiphile Solutions on the Sol−Gel Transitions, the Gelation Speed, and the Gel Characteristics Takahiro Otsuka, Tomoki Maeda, and Atsushi Hotta* Department of Mechanical Engineering, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan S Supporting Information *

ABSTRACT: Hydrogels made of peptide amphiphiles (PA) have attracted a lot of interest in biomedical fields. Considering the applications of PA hydrogels, the control of the gelation speed and the gel characteristics is essential to predominantly determine the usefulness and practicability of the hydrogels. In this work, the effects of the salt concentrations using sodium dihydrogenorthophosphate (NaH2PO4) on the sol−gel transition behaviors, especially the gelation speed and the gel characteristics of the designed PA (C16−W3K) hydrogels in aqueous solution were discussed. It was found that the original solution state before rheological testing was independent of the salt concentration, which was confirmed by observing the self-assembly structures and the peptide secondary structures of PA through transmission electron microscopy (TEM) and circular dichroism spectroscopy (CD). The PA solutions with different salt concentrations, however, presented a profound difference in the gelation speed and the gel characteristics: the solution exhibited higher gelation speeds and higher mechanical properties at higher salt concentrations. Concurrently, the density, the length of wormlike micelles, and the conformational ratio of β-sheets to α-helices in the equilibrium PA solutions all increased with the increase in the salt concentrations.

1. INTRODUCTION A peptide amphiphile (PA) is a surfactant agent composed of hydrophilic and hydrophobic molecular segments.1,2 The used PA in our experiments was a block copolymer with a hydrophilic peptide coupled to a hydrophobic alkyl tail. Due to the hydrogen bondings in the peptide block, PA could establish several unique secondary conformational structures of α-helices and β-sheets as well as spherical and wormlike micelle structures.3,4 Furthermore, PA could change its solution states from sol to gel, while simultaneously transforming their secondary and micelle structures from α-helix to β-sheet, and from spherical to wormlike, respectively. Other PAs also showed such sol−gel transitions by entangled nanofibers formed by rodlike micelles or nanoribbons.4−7 Because of the biocompatibility and the narrow distribution of the molecular weight of PA after synthesis, PA is expected to be utilized for biomedical applications such as tissue-engineering matrix by controlling the multiscale structural transitions mentioned above.8 The multiscale structures of PA were directly affected by several controllable external factors such as PA concentrations,9,10 temperature,7,10 pH,10−12 the length of hydrophobic segments,7,12,13 and the salt concentration,14−19 and the concurrent structural transitions were also reported. The studies of the salt-concentration effects on PA solutions are biologically highly significant, as human cells consist essentially of inorganic salts, proteins, and water, where inorganic © 2014 American Chemical Society

substances were bound to proteins to support the vital functions of the protein molecules in the human body. It is indeed recognized that inorganic salts have huge effects on peptides mainly because the salts present electrostatic screening.20,21 The electrostatic screening induced by salts can modify the microstructures of the micelles and the molecular conformations, eventually changing the sol−gel states of the solution. As a matter of course, inorganic salts can also modify the multiscale structural transitions of the peptides, especially changing the gelation speed and the gel characteristics. Structural modifications of the peptides by adding salts were reported by several groups.15,17,22−26 Ghoorchian et al. reported that the size of micellar particles of a three-armed star elastinlike polypeptide could be controlled by adjusting NaCl concentrations: the particle diameters at the salt concentrations below 15 mM, between 15 mM and 45 mM, and above 45 mM showed 30 nm or smaller (which grew linearly by increasing the salt concentrations up to 15 mM), approximately constant in the range of 60−65 nm, and about 360 nm, respectively.23 Castelletto et al. reported that the high concentrations of NaCl (>150 mM) induced a transition of secondary structures from random coils to β-sheets for the peptide with the chemical sequence of NH2-βAla-Ala-Lys-Leu-Val-Phe-Phe-COOH.15 Received: March 31, 2014 Revised: August 9, 2014 Published: September 8, 2014 11537

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acid chain composed of 13 alanines (A) with a tryptophan (W) (for the fluorescence measurements of the PA concentration), and three spatially separated lysines (K) (for the enhancement of solubility in water) attached to a 16-carbon alkyl tail (Figure 1). Pure water as solvent was obtained from Wako Pure Chemical Industries, Ltd. Sodium dihydrogenorthophosphate (NaH2PO4) was obtained from Junsei Chemical Co., Ltd. NaH2PO4 was selected as salt, which is the most commonly used salt for buffer solution that carries out effective buffer action to retain pH of cell fluid. All reagents were of analytical grade and were used as received. NaH2PO4 was added into pure water at the concentration of 0, 0.05, 0.5, and 5 mM, and then C16−W3K was put into each solvent in order to make PA solution with 80 μM of PA concentration at 25 °C. C16−W3K was dissolved instantly in the prepared solvent without stirring. All experiments were conducted in the same environment to prevent any sorts of shear history before the experiments. 2.2. Rheological Measurements. Steady and dynamic rheological measurements were performed by rheometer (ARES-G2, TA Instruments Japan Inc.) using a cone and plate geometry with 50 mm in diameter and the cone angle of 0.04 rad. A Peltier-based temperature control device was used to set the temperature at 20 °C. The gap between the cone plate and the Peltier plate was kept constant at 0.05 mm. The sample volume was 1.4 mL. The viscosity of the C16−W3K solution prepared immediately after making solution was measured by changing the shear rates from 1 to 1000 s−1 with steady flow. The measurements were repeated with the same sample in the same fashion until the sample reached its equilibrium state. “One cycle” was defined as one shear-rate scan from 1 to 1000 s−1. The storage modulus G′ and the loss modulus G″ were determined using an oscillatory frequency sweep changing the angular frequency from 0.1 to 200 rad/s. The dynamic mechanical analysis (DMA) of the samples before and after viscosity testing was performed within the linear viscoelastic region. The sol and gel states were detected by observing the storage modulus (G′) and the loss modulus (G″): G′ < G″ for the sol state and G′ > G″ for the gel state. 2.3. Transmission Electron Microscopy. TEM images were obtained by TECNAI SPIRIT (FEI Company) with an accelerating voltage of 120 kV to analyze the self-assembled structures of C16−W3K in the solvents with different salt concentrations before and after the viscosity testing. 40 μL of PA solution was deposited on carbon grids without staining, which was immediately mounted on a sample holder of TEM for the observation. 2.4. Circular Dichroism Spectroscopy. Ellipsometric studies on C16−W3K solutions were conducted by a Chirascan CD spectrometer (Applied Photophysics Ltd.) measuring through 1 mm optical path length to analyze the secondary structures of the peptide W3K in the C16−W3K. The CD spectrometer have a high sensitivity by means of small noise of polarization with two polarization prisms which other CD spectrometer has one, therefore it can measure the vacuumultraviolet region accurately. The experiment was carried out under the nitrogen flow rate of 5 L/min. The CD spectra of the C16−W3K solutions with different salt concentrations before and after the viscosity testing were measured through the wavelengths ranging from 190 to 260 nm at 0.5 nm intervals at 20 °C. All presented CD spectra were the average of three CD scans subtracting all the background signals of the cell and solvents from the original CD value.

Regarding the gelation speed of peptides or protein of living organisms by adding salts, Fujiwara et al. reported that the gelation of hen egg white lysozyme, which is one of the biological proteins, was accelerated by NaCl.27 In another example of bioprotein, Otte et al. also found that the gelation of a whey-protein isolate solution was accelerated by CaCl2 concentration, temperature, enzyme concentration, and protein concentration.28 Ozbas et al. reported that NaCl induced the transition from random coils to β-sheets of the MAX1 peptide and increased the gelation speed of the peptide solution.29 In order to systematically analyze the effects and the mechanisms, a careful investigation of salt on PA with a simplified chemical structure with an appropriate gelation speed has been desired. There have only been a few reports regarding the salt effects on the mechanical characteristics of peptide gels. Dagdas et al. reported that CaCl2 hardened the gel of the PA with the VVAGRGD sequence attached to a lauric acid.24 Kuang et al. reported that the hardness of the gel that was made from pentapeptide−epitope hydrogelator solution could be transmuted by K+ concentrations due to the change in the network structures: the hardest gel could be obtained at 8.38 mM of K+ concentration due to the nanofiber aggregation forming multiple cross-linkings.30 Here in this paper, the effects of salt (NaH 2 PO 4) concentrations on the gelation speed due to the hierarchical structural transitions of a designed PA called C16−W3K were carefully investigated. It has already been reported that C16− W3K showed multiscale structural transitions with time,31 temperature,32,33 and mechanical shear.34 Each parameter was independent, and the aqueous C16−W3K solution could change the micellar assemblies, the molecular conformations, and the solution state, from spherical to wormlike, from α-helix to β-sheet, and eventually from sol to gel states, respectively.31−34 Effects of the salt concentration on the structural transitions of PA have never been studied in depth. The studies of the salt-concentration effects on PA solutions are biologically highly significant, since living cells including human cells unexceptionally consist of inorganic salts, proteins, and water, where inorganic substances were essentially linked with proteins to support the vital biological functions of the protein molecules in the living organism. The effects of salt on the sol− gel transition rates of PA solution by observing the multiscale structures were first examined by rheological measurements, transmission electron microscopy (TEM), and circular dichroism spectroscopy (CD).

2. EXPERIMENTS 2.1. Materials and Sample Preparations. The peptide amphiphile C16−W3K (Figure 1) with the molar mass of Mn ∼ 1751 as a solute was synthesized by Scrum Inc. in the highest purity available (95%). The C16−W3K molecule is a 17-amino

Figure 1. Chemical structure of the PA conjugates. 11538

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Figure 2. Viscosity measurements of the C16−W3K solutions with different salt concentrations: (a) 0, (b) 0.05, (c) 0.5, and (d) 5 mM.

The final viscosity value at equilibrium and the requisite shear cycles to reach the equilibrium viscosity varied with samples with different salt concentrations: the viscosity became 1.8 × 10−1 Pa s after the 21st shear cycles for the 0.05 mM sample (Figure 2b), 2.8 × 10−1 Pa s after the fifth cycle for 0.5 mM (Figure 2c), and 5.7 × 10−1 Pa s after the fourth cycle for the 5 mM sample (Figure 2d), measured at the shear rate of 10° s−1. The viscosity result of PA solution at the physiological salt concentration of 150 mM was presented in Figure S1 of the Supporting Information. It was found that the gelation speed of the solution with 150 mM salt concentration was as fast as that of 5 mM (fourth cycle), clearly indicating that the rapid increase in the gelation speed was completed by 5 mM. The final viscosity value at the shear rate of 10° s−1 became higher with the increase in the salt concentrations, indicating that a firmer network structure of C16−W3K could be attained at higher salt concentrations. Additionally, as the salt concentration increased, the number of cycles required for C16−W3K solutions to reach equilibrium states decreased (Figure 3). The viscosity of 0 mM C16−W3K solution increased gradually with the viscosity testing cycles (i.e., slow gelation), while that of 5 mM C16−W3K solution increased rapidly with the increase in the viscosity testing cycles (i.e., fast gelation). Our previous works reported that temperature and the mechanical shear during the viscosity testing could induce and accelerate the gelation of C16−W3K solutions with the structural transitions from spherical micelles to wormlike micelles as well as with the molecular conformational transitions from α-helix to β-sheet.31 It was, therefore, suggested from these new experimental results that, at low salt concentrations, spherical micelles gradually transformed into wormlike micelles, while α-helix conformations transformed to β-sheet conformations rather progressively, whereas at high salt concentrations, these transitions occurred relatively fast.

2.5. Infrared Spectroscopy. Fourier transform infrared spectroscopy (FTIR) analysis was carried out using FTIR-4200 (JASCO) with CaF2 glass to identify the secondary structures of the peptide W3K. The IR spectra of the C16−W3K solutions with different salt concentrations before and after the viscosity testing were measured through the wavenumbers ranging from 1750 to 1550 cm−1, and the wavenumber resolution was 4.0 cm−1. 200 mL of the C16−W3K solution specimen for the analysis was dropped on a CaF2 glass and dried for 1 h in a vacuum oven at 25 °C. All presented IR spectra were the average of 100 scans. All spectra were normalized at 1655 cm−1 at the α-helix peak.

3. RESULTS AND DISCUSSION 3.1. Enhancement of the Gelation Speed by Increasing the Salt Concentrations. The viscosity measurements were carried out to analyze the mechanical properties of C16− W3K solutions at different salt concentrations. Figure 2 shows the viscosity results of C16−W3K solutions with different salt concentrations. The viscosity of the C16−W3K solution with 0 mM of the salt concentration (i.e., pure water) was between 10−3 and 10−2 Pa s throughout the whole range of shear rates, which was observed up to the 19th shear cycle. The viscosity characteristics were very similar to the characteristics of Newtonian fluid such as pure water without C16−W3K. After the 20th cycle, the viscosity began to climb up, which even became up to 1.1 × 10−1 Pa s after the 25th cycle, nearly 100 times higher than the original viscosity at the shear rate of 10° s−1. At the same time, after transition, a shear-thinning characteristic appeared in viscosity, indicating a possible network-formation of entangled thin fibers in the solution.35 The viscosity results of other samples with different salt concentrations also showed a similar trend in viscosity with the shear-thinning characteristics after transition. 11539

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sufficient viscosity testing became much higher by 100−1000 times than those before the viscosity testing. In addition, it was also found that G′ and G″ were essentially independent of frequency and that there was no crossover detected throughout the whole range of frequency. Moreover, the C16−W3K solutions with higher salt concentrations possessed higher storage moduli after transition, where G′ were 9.4, 12.1, 15.4, and 31.7 Pa at 10 rad/s for the gel specimens with the salt concentrations of 0, 0.05, 0.5, and 5 mM, respectively. The fairly strong solidlike features of DMA signals for the C16− W3K samples after viscosity testing demonstrated reasonably firm cross-linked networks of wormlike micelles established in C16−W3K gels, especially at higher salt concentrations.36 To summarize, the sol−gel transition speed of C16−W3K solutions under the same external conditions became significantly faster with the increase in the salt concentration of C16−W3K solutions. Considering the simultaneous multiscale structural transitions previously reported by our group and others, it is strongly suggested that the spherical to wormlike micelle-transition speed of C16−W3K as well as the concomitant α-helix to β-sheet conformational-transition speed should be both enhanced, as the salt concentration increased in the C16−W3K solutions. 3.3. Advancement in the PA Structural Transitions by Increasing the Salt Concentrations. In order to confirm the self-assembled microstructures such as spherical and wormlike micelles and thin-fiber networks mentioned above, TEM micrographs were taken for C16−W3K specimens (Figures 6 and 7 and Figure S2 of the Supporting Information). Figure 6 and Figure S2 of the Supporting Information show the TEM micrographs of C16−W3K samples before the viscosity testing, when all solutions with different salt concentrations were entirely in the sol-state. Spherical micelles and a few aggregates

Figure 3. Number of the viscosity testing cycles necessary to reach equilibrium viscosity as a function of the salt concentration. The line presents the number of the cycles of the PA sample without salt to reach equilibrium viscosity.

3.2. Increase in the Hardness of PA Gels with the Addition of Salt. DMA measurements were performed to confirm the sol or the gel state of C16−W3K solutions before and after the structural transition by viscosity testing (Figures 4 and 5). Prior to the viscosity testing, a series of DMA measurements were performed for the C16−W3K solutions with different salt concentrations. It was found that G′ of all samples were lower than G″ throughout the whole frequency range of the DMA measurements, indicating that all C16−W3K solutions were originally in the liquid state (sol state). In contrast, after the sufficient cycles of viscosity testing, G′ of the samples became higher than G″ at each salt concentration for the whole frequency range, indicating that the C16−W3K solutions became gel after the sufficient cycles of the viscosity testing. In fact, both G′ and G″ of the samples after the

Figure 4. DMA results of the C16−W3K solutions before viscosity testing (i.e., before transition) with different salt concentrations: (a) 0, (b) 0.05, (c) 0.5, and (d) 5 mM. 11540

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Figure 5. DMA results of the C16−W3K solutions after the viscosity testing (i.e., after transition) with different salt concentrations: (a) 0, (b) 0.05, (c) 0.5, and (d) 5 mM.

Figure 6. TEM micrographs of C16−W3K solutions before viscosity testing (i.e., before structural transitions) at two different salt concentrations: (a) 0 and (b) 5 mM. These scale bars are 500 nm. TEM micrographs at other different salt concentrations are presented in Figure S2 of the Supporting Information.

of the spherical micelles were observed, whereas wormlike micelles were not recognized in each C16−W3K specimen. Shimada et al. reported that the diameter of a spherical micelle of C16−W3K was ∼5 nm studied by small-angle neutron scattering (SANS).37 We observed many spherical micelles and a few aggregates of the spherical micelles by an atomic force microscopy (AFM) presented in Figure S3 of the Supporting Information. The diameter of the micelles was confirmed to be around 5 nm by AFM. In fact, the micelle structures observed for the C16−W3K solutions before the viscosity testing were almost the same regardless of the difference in the salt concentrations. As was expected, the TEM micrographs of the C16−W3K solutions after the viscosity testing, when the solutions were in the gel state, exhibited wormlike micelles with a width of about 5 nm and a length of microns. The TEM results were in good agreement with the previous results of TEM studies on the same PA specimens but in a different

Figure 7. TEM micrographs of C16−W3K specimens after viscosity testing (i.e., after structural transitions) at different salt concentrations: (a) 0, (b) 0.05, (c) 0.5, and (d) 5 mM. These scale bars are 500 nm.

buffer composition (10 mM sodium chloride with 1 mM sodium phosphate).31 The TEM micrograph of 0 mM C16− W3K solution (i.e., without salt) presented significantly shorter lengths of fibers than the C16−W3K in buffer solutions (i.e., with salt) (Figure 7). Actually, the number of wormlike micelles, hence the density of the wormlike micelles, increased as salt concentration increased. It was considered from all these 11541

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which distinctly represented a clear β-sheet peak after viscosity testing. It was therefore concluded that as the salt concentration increased, the intensity of α-helix peaks at 204 and 222 nm became weaker while that of a β-sheet peak at 216 nm became stronger, indicating a clear structural transition from α-helix to β-sheet. Figure 9 shows FTIR spectra of C16−W3K specimens with different salt concentrations (a) before and (b) after viscosity

experimental results that the mechanical properties of C16− W3K solutions were largely attributed to the density and the length of the wormlike micelles: the C16−W3K solutions became harder with the increase in the density and the length of the wormlike micelles. It was therefore concluded that the density and the length of the wormlike micelles in the C16− W3K solutions could be effectively controlled by the salt concentration in the buffer. 3.4. Progress in the Conformational Transitions of Peptide Secondary Structures by Adding Salt. CD and FTIR measurements were performed to analyze the secondary structures of the peptide in C16−W3K. Figure 8 presents the

Figure 9. FTIR spectra of C16−W3K solutions with different salt concentrations (a) before and (b) after viscosity testing.

testing. The FTIR peak at 1625 and 1655 cm−1 were generally assigned to the CO stretches (amide I band) of the β-sheet and the α-helix secondary structures in polypeptides, respectively.31,40 In Figure 9a, all C16−W3K solutions with different salt concentrations before the viscosity testing had a single FTIR peak at 1655 cm−1, which indicated the major existence of α-helix conformations. The FTIR spectra of the specimens after the viscosity testing, on the other hand, showed a gradual increase in the FTIR intensity at the peak of 1625 cm−1, while also presenting a gradual decrease at 1655 cm−1, with the increase in the salt concentration (Figure 9b). The results, therefore, indicated that the α-helix conformations in W3K have efficiently transformed into β-sheet conformations during the viscosity testing and that the existence of salt has indeed increased the ratio of β-sheet conformations at equilibrium. Here again, it was confirmed that the salt played an important role in the determination of the conformational ratios of α-helix to β-sheet. To summarize, the peptide W3K of C16−W3K solutions established α-helix conformations before viscosity testing, whereas after the testing, the α-helix conformations transformed to β-sheet conformations. The ratios of the structural transition increased as the salt concentration increased. Considering the experimental results described in Enhancement of the Gelation Speed by Increasing the Salt

Figure 8. CD spectra of C16−W3K solutions with different salt concentrations (a) before and (b) after viscosity testing.

CD spectra of C16−W3K solutions with different salt concentrations (a) before (i.e., sol) and (b) after (i.e., gel) viscosity testing. Generally, α-helices have negative Cotton effects at the wavelengths of ∼208 nm and ∼222 nm and a positive Cotton effect at around 192 nm, while β-sheets show a negative Cotton effect at around 216 nm and a positive Cotton effect at around 197 nm.38,39 Here the Cotton effect indicates the characteristic change in the circular dichroism in the vicinity of an absorption band of a test substance. All C16−W3K solutions with different salt concentrations before the viscosity testing presented two negative bands at the wavelengths of 204 and 222 nm in CD, which demonstrated that W3K predominantly possessed α-helix conformations (Figure 8a). However, with the increase in the salt concentration, the negative band at the wavelength of 204 nm of a typical α-helix peak, shifted to higher wavelengths gradually after the viscosity testing (Figure 8b) (please refer to Figures S4−S7 of the Supporting Information for more details of the CD spectra of the C16−W3K solutions before and after viscosity testing at each salt concentration). Eventually, the specimen of 5 mM salt concentration exhibited one single negative band at 216 nm, 11542

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injectable gels and artificial extracellular matrices in the tissue engineering.

Concentrations, Increase in the Hardness of PA Gels with the Addition of Salt, and Advancement in the PA Structural Transitions by Increasing the Salt Concentrations, salt effectively promoted the multiscale structural transitions of C16−W3K by eventually enhancing the gel fraction and gelation speed of the aqueous C16−W3K specimens. Our previous research exhibited that the hierarchical structural transitions were accelerated by increasing temperature or imposing mechanical shear. In this research, the effects of the salt concentrations of the PA solution up to the physiological salt concentration of 150 mM on the structural transitions of PA solution were studied. It was found that the transition speed was dramatically increased at surprisingly very low concentration. Actually the substantial increase was only observed up to the concentration of 5 mM, which was well below 150 mM. The results indicated that the salt concentration was another important parameter that significantly affected the multiscale structures of PA, including the density and length of the wormlike micelles, the conformational ratios of β-sheets to αhelices, and the hardness of the resulting PA gels. It was known that the intermolecular hydrogen bonding in the β-sheet structures of C16−W3K caused the structural stabilization of wormlike micelles.6,34,41 Therefore, it was fairly understandable that the higher ratios of β-sheet to α-helix in the C16−W3K specimens caused by the higher salt concentration could increase the density and length of wormlike micelles, allowing longer wormlike micelles to form to produce more entanglements in the aqueous C16−W3K gels, which eventually presented the faster gelation and higher mechanical characteristics of the resulting gels. The differences in the gelation speeds and the gel characteristics at equilibrium could be highly attributed to the effects of electrostatic screening of salt. It is known that the electrostatic screening of salt reduces electrostatic repulsion of peptides or proteins, inducing straightforward aggregations of peptide or protein molecules.20,21,27,42,43 In fact, Dong et al. reported that increasing NaCl concentration resulted in a highly viscous solution with a dramatic increase in fiber length due to the electrostatic screening.18 Moreover, as for the formation process of the wormlike micelles of C16−W3K, Shimada et al. demonstrated subsequent micelle-chain elongation of C16−W3K by adding spherical micelles to the end of growing cylindrical micelles to form wormlike micelles in a process mimicking the chain-growth polymerization.37 They considered that, during the formation of wormlike micelles, the electrostatic screening of the salt could reduce the electrostatic repulsion of W3K due to the lysines in C16−W3K. The lowering of the electrostatic repulsion therefore induced spherical micelles to assemble, which resulted in the formation of wormlike micelles, eventually presenting faster hierarchical structural transitions in aqueous C16−W3K solutions. The faster hierarchical structural transitions from sol to gel states, from spherical to wormlike micelles, and from α-helix to β-sheet conformations, were first controlled just by salt concentrations in this work without using other factors such as pH and peptide concentrations: Chen et al. reported that the ratios of β-sheets of the naphthalene-dipeptide and the gelation speed of the solution increased as pH decreased;44 Veerman et al. also reported that the ratios of β-sheets of the peptide called MAX1 and the hardness and the gelation speed of the peptide solution increased with the increase in the peptide concentration.9 The control of the hierarchical structural transitions solely by salt is expected to be applicable to biomaterials such as

4. CONCLUSIONS A simple process by adding salt to promote the gelation speed and the multiscale structural transitions of a peptide amphiphile solution was introduced. The gelation behavior of the solution was analyzed by the viscosity measurement by controlling the transition speeds of the C16−W3K solution. It was demonstrated that the gelation speed became faster with the increase in salt concentrations. It was also found by DMA that the gel became harder with the increase in the salt concentration. TEM images showed that the micelle structures of sol specimens were spherical micelles regardless of the salt concentration, while those of gel specimens were wormlike micelles with increasing density and length by increasing the salt concentration. It was found that the enhancement of the mechanical property of C16−W3K solution resulted from the increasing density and length of wormlike micelles caused by the increase in salt concentration. It was considered that the salt facilitated the α-helix to β-sheet transition in the W3K peptide due to the electrostatic screening of salt examined by CD and FTIR measurements. It was found that the salt concentration was a newly found important parameter and the only parameter that could induce the multiscale structures of C16−W3K as well as significantly change the final equilibrium values of the density and the length of the wormlike micelles, the conformational ratios of β-sheets to α-helices, and the hardness of the resulting PA gels, which had not been observed by using other parameters such as time, temperature, and mechanical shear.

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ASSOCIATED CONTENT

S Supporting Information *

Viscosity results of the C16−W3K solution with 150 mM of salt concentration in Figure S1, TEM images of the C16−W3K solutions with the salt concentrations of 0.05 mM and 0.5 mM before viscosity testing shown in Figure S2, an AFM image of the C16−W3K solutions with 5 mM of the salt concentration before the structural transition in Figure S3, and the CD spectra of the C16−W3K solutions before and after viscosity testing at each salt concentration shown in Figure S4−S7. 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]. Tel: +81 455661604. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS: “KAKENHI”) (Grant 23360294 to A.H.), a Grant-in-Aid for Scientific Research (S) (Grant 21226006 to A.H.), a Grant-in-Aid for Scientific Research for Challenging Exploratory Research (Grant 24656395 to A.H.). We appreciated the fruitful discussion about PA with Prof. Matthew Tirrell. 11543

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