Coassembly of Oppositely Charged Short Peptides into Well-Defined

Jan 29, 2010 - Triisopropylsilane (TIS) was purchased from ACROS (USA) and used without further purification. All other reagents and solvents are of ...
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J. Phys. Chem. B 2010, 114, 2365–2372

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Coassembly of Oppositely Charged Short Peptides into Well-Defined Supramolecular Hydrogels Xiao-Ding Xu, Chang-Sheng Chen, Bo Lu, Si-Xue Cheng, Xian-Zheng Zhang,* and Ren-Xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan UniVersity, Wuhan 430072, P. R. China ReceiVed: October 27, 2009; ReVised Manuscript ReceiVed: January 6, 2010

Two types of oppositely charged short peptides comprised of a hydrophobic N-fluorenyl-9-methoxycarbonyl (FMOC) tail and a peptide backbone were designed and prepared via a standard solid phase peptide (SPPS) technique. When mixing these two oppositely charged peptides in water at a neutral pH, a supramolecular hydrogel with fibroid morphology could be formed via the electrostatic attraction triggered coassembly. The spectroscopic techniques indicated that the hydrogen bonding interactions of the peptide backbones resulted in the formation of antiparallel β-sheet like superstructure, and the fluorenyl rings connected to the peptide backbones were thus π-stacked with each other through an antiparallel fashion in the formed nanofibers. Due to the weak flexibility of peptide chains and steric hindrance of rigid fluorenyl rings during the initial process of the coassembly of the oppositely charged peptides, a relatively slow self-assembly was presented, and a higher concentration of the oppositely charged peptides was necessary for this supramolecular hydrogel formation. The strategy demonstrated in this study can be developed as a convenient approach for different types of short peptides to coassemble into a supramolecular hydrogel with multiple functions for the biomedical applications. Introduction Arising from the abundant examples of protein self-assembly existing in nature, much recent attention has been drawn to create a new generation of biomaterials based on the selfassembly of peptides and their derivatives. By exploiting the spontaneous or induced molecular arrangement upon external stimuli such as pH and/or temperature, peptides and their derivatives are able to grow from homogeneous solution into fibroid structure in water via noncovalent forces including π-stacking, hydrogen bonding, and hydrophobic interactions.1-6 The subsequent entanglement of fibers or network structure formation and trapping solvent via surface tension can generate supramolecular hydrogels.7-10 Due to their advantages over the common polymeric hydrogels such as without introduction of additional covalent cross-linker to result in possible loss of the biocompatibility and biodegradability of hydrogelators, these highly hydrated scaffolds present a potential alternative in tissue engineering.11-14 To date, a variety of structural motifs such as coiled coils,15 β-sheets,16 β-hairpins,17 and peptide amphiphiles (PAs)18 have been established for the self-assembly of peptides and their derivatives with long peptide backbones (generally composed of at least ten amino acid residues). Since the first report of an aromatic dipeptide which can self-assemble into a nanotube,19 hollow sphere,20 and nanofiber,21 a relative new class of supramolecular hydrogels resulted from the self-assembly of much shorter peptides with aromatic tails such as the popular N-fluorenyl-9-methoxycarbonyl (FMOC) group which has been recently developed.22-25 In these systems, the aromatic moieties play a key role in the self-assembly process through π-stacking, while the peptide backbones stabilize via hydrogen bonding * Corresponding author. Tel./fax: +86-27-68754509. E-mail address: [email protected].

interactions to form β-sheet like architectures. Up to now, the self-assembly of numerous FMOC-based shorter peptides composed of different amino acid residues including serine, threonine, glycine, alanine, leucine, and phenylalanine has been extensively studied.22,23 In general, the self-assembly of most peptides, especially the peptide loading charges, requires the external stimuli such as altering the solution pH22,23 or addition of exogenous salt.13,17 Because these charged peptides can be dissolved in aqueous solution via the intermolecular electrostatic repulsion, the addition of exogenous salt or altering the solution pH can screen the charge repulsion or accomplish deprotonation to obtain an expected self-assembly. Ulijin et al.26 recently reported that the self-assembly of FMOC-diphenylalanine can result in a structural behavior transition from ionized molecules to paired fibrils and even large rigid ribbons upon the decrease of solution pH. Actually, besides altering solution pH or introduction of exogenous salt to trigger the self-assembly of peptides, Stupp et al.27,28 and Yu et al.29,30 recently demonstrated that simply mixed oppositely charged peptides can quickly self-assemble into nanofibers with β-sheet architectures via intermolecular electrostatic attraction. In comparison with the traditional pH triggered self-assembly of peptides, this new convenient strategy can be administrated at a neutral pH and provides a potential to combine two bioactive signals within a single nanofiber,27 presenting superiority in the application of tissue engineering and 3D cell culture. In these systems, the peptides and their derivatives usually have no29,30 or flexible hydrophobic tails.27,28 Besides, the peptide backbones are generally composed of peptide components exceeding ten amino acid residues in length. Therefore, the flexibility of molecular chains would be in favor of the occurrence of electrostatic attraction of oppositely charged peptides. The main focus of this work was to investigate the influence of electrostatic attraction on the self-assembly of

10.1021/jp9102417  2010 American Chemical Society Published on Web 01/29/2010

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SCHEME 1: Molecular Structures of Peptides 1-3 Used for Self-Assembly

oppositely charged short peptides with rigid aromatic tails. On the basis of this point, two types of oppositely charged FMOCbased short peptides were designed and synthesized. When mixing these two types of oppositely charged peptides in water at a neutral pH, a supramolecular hydrogel with fibroid network structure could be formed via the electrostatic attraction triggered coassembly. The spectroscopic techniques demonstrated that the peptide backbones carried out an antiparallel β-sheet like arrangement, and the fluorenyl rings were thus positioned to π-stack with each other through an antiparallel fashion in the formed nanofibers. Because of the weak flexibility of peptide chains and steric hindrance of rigid fluorenyl rings during the initial process of the coassembly of the oppositely charged peptides, a relatively slow self-assembly was presented, and a higher concentration of the oppositely charged peptides was necessary for gelation. Experimental Methods Materials. N-Fluorenyl-9-methoxycarbonyl (FMOC) protected L-amino acids (FMOC-Gly-OH, FMOC-Asp(OtBu)-OH, FMOC-Arg(Pbf)-OH, FMOC-Val-OH, FMOC-Lys(Trt)-OH) and 2-chlorotrityl chloride resin (100-200 mesh, loading: 1.32 mmol/g) were purchased from GL Biochem (Shanghai) Ltd. (China) and used as received. N-Hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA), o-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), and piperdine were provided by Shanghai Reagent Chemical Co. (China) and used directly. Dimethylformamide (DMF) and diisopropylethylamine (DiEA) were obtained from Shanghai Reagent Chemical Co. (China) and distilled prior to use. Triisopropylsilane (TIS) was purchased from ACROS (USA) and used without further purification. All other reagents and solvents are of analytical grade and used directly. Synthesis of Peptides. All the peptides (Scheme 1) were synthesized manually in 1.98 mmol scale on the 2-chlorotrityl chloride resin employing a standard FMOC solid-phase peptide

Xu et al. synthesis (SPPS) technique. Before the synthesis, the resin was washed with CH2Cl2 (three times) and DMF (three times) and then immersed in DMF for 30 min. After draining off DMF solution, a DMF solution of the mixture of FMOC protected amino acid (4 equiv relative to resin loading) and DiEA (6 equiv) was added to the resin and shaken for 2 h at room temperature. After removing the reaction solution, the resin was washed with DMF (three times). Subsequently, 20% piperidine/ DMF (V/V) solution was introduced to the resin to remove the FMOC protected groups. After shaking for 30 min at room temperature, the reaction solution was drained off, and the resin was washed with DMF (three times). The presence of free amino groups was indicated by a blue color in the Kaiser test. Thereafter, a DMF solution of the mixture of FMOC protected amino acid (4 equiv), HBTU (4 equiv), HOBt (4 equiv), and DiEA (6 equiv) was added. After shaking for 1.5 h at room temperature, the reaction solution was drained off, and the resin was washed with DMF (three times). The absence of free amino groups was indicated by a yellow color in the Kaiser test. After repetition of the deprotection and acylation reaction, the resin was finally washed with DMF (three times) and CH2Cl2 (three times) and dried under vacuum for 24 h. Cleavage of the expected peptide and the removal of the protected groups of side chains from the dried resin were performed using a mixture of TFA, deionized water, and TIS in the ratio of 95:2.5:2.5. After 2 h shaking at room temperature, the cleavage mixture and three subsequent TFA washings were collected. The combined solution was concentrated to a viscous solution by rotary evaporation. Cold ether was added to precipitate the product. After washing with cold ether (five times) to remove TFA residual, the precipitate was dissolved in distilled water and then freeze-dried under vacuum for 3 days. The purity of the products was examined by high-pressure liquid chromatography (HPLC) with a C18 column and using a linear gradient of acetonitrile and DI water containing 0.1% TFA. Peptide 1: purity, 95% by HPLC; IR, ∼3470 cm-1 amide A band, ∼1657 cm-1 amide I band, ∼1558 cm-1 amide II band; MS, calculated 766.4, [M + H]+ found 767.3. Peptide 2: purity, 92% by HPLC; IR, ∼3470 cm-1 amide A band; ∼1654 cm-1 amide I band, ∼1557 cm-1 amide II band; MS, calculated 682.3, [M + H]+ found 683.3. Peptide 3: purity, 94% by HPLC; IR, ∼3450 cm-1 amide A band, ∼1655 cm-1 amide I band, ∼1558 cm-1 amide II band; MS, calculated 952.5, [M + H]+ found 953.5. Acid-Base Titration. The solution of peptides 1, 2, and 3 was prepared in distilled water at a concentration of 2 mg/mL. For the pKa titrations of acidic peptides 1 and 2, 0.01 M NaOH aqueous solution was added in 1-5 µL increments, starting at a low pH of 3, whereas for the pKa titration of basic peptide 3, 0.01 M HCl aqueous solution was added in 1-5 µL increments, starting at a high pH of 11. After each addition, the sample was constantly stirred for 5 min at room temperature, and the pH value of the solution was measured using a PB-10 Sartorius pH meter. pH Triggered Self-Assembly. A solution of peptides 1 and 2 was prepared in distilled water at a pH of 11 with a concentration of 5 mg/mL. Then, concentrated HCl was added in 1-5 µL increments, and the corresponding solution pH was measured on a PB-10 Sartorius pH meter after shaking the solution for 5 min. When the solution pH decreased to a value of 3.8, well-defined supramolecular hydrogels were formed. To investigate the self-assembly behavior of peptide 3, the aqueous solution with a concentration of 5 mg/mL was prepared at a pH of 4. Then, concentrated NaOH was added in 1-5 µL

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Figure 1. Optical and TEM images of the supramolecular hydrogels self-assembled from peptides 1 (Gel1) and 2 (Gel2) at a pH of 3.8.

increments. The solution pH was subsequently measured on a PB-10 Sartorius pH meter after shaking the solution for 5 min. Electrostatic Attraction Triggered Coassembly. Different amounts of peptide 1 or 2 were mixed with peptide 3 in distilled water. The obtained peptide solution was adjusted to a neutral pH by the addition of concentrated HCl or NaOH aqueous solution and then placed at room temperature to carry out gelation. Oscillatory Rheology. The solution of peptides 1 and 2 was prepared as described above and transferred to the ARES-RFS III rheometer. After decreasing the solution pH to a value of 3.8, the storage modulus (G′) and loss modulus (G′′) were recorded to understand the viscoelastic properties of the supramolecular hydrogels. As for the supramolecular hydrogels resulting from the coassembly of the oppositely charged peptides, due to the relatively long gelation time (around 12 h), their viscoelastic properties were studied after the hydrogel formation. Transmission Electron Microscopy (TEM). The supramolecular hydrogels self-assembled from peptides 1 and 2 were diluted in distilled water at a pH of 3.8, and a small volume of diluted hydrogel solution was applied to a copper grid with Formvar film and dried before the observations on a Tecnai G20 S-TWIN transmission electron microscope (TEM). The morphology of the supramolecular hydrogels self-assembled from the oppositely charged peptides was studied similarly by diluting the hydrogels in distilled water at a neutral pH. Circular Dichroism. The supramolecular hydrogels were fixed in a 0.5 mm quartz cell and analyzed on a Jasco J-810 spectropolarimeter with 4 s accumulations every 1 nm and averaged over five acquisitions. Fourier Transform Infrared Spectroscopy (FT-IR). FTIR spectra of the supramolecular hydrogels were collected on a Perkin-Elmer spectrophotometer by pressing the lyophilized hydrogel samples into KBr pellets. Wide-Angle X-ray Diffraction (WXRD). The X-ray diffraction patterns were obtained by a Shimadzu XRD-6000 diffractometer with a Ni filter and Cu KR1 (λ ) 1.54056 Å, voltage ) 40 kV; current ) 40 mA). Before the scanning, the supramolecular hydrogels were spread on glass slides and allowed to air-dry. Fluorescence Spectroscopy. Fluorescence emission spectra of the supramolecular hydrogels and the solution of the peptides were recorded on a LS55 luminescence spectrometer (PerkinElmer) with excitation at 265 nm and emission data ranging between 300 and 700 nm.

Figure 2. Oscillatory rheology of the supramolecular hydrogels selfassembled from peptides 1 (Gel1) and 2 (Gel2) at a pH of 3.8.

Results and Discussion pH Triggered Self-Assembly. The chemical structures of the prepared peptides are shown in Scheme 1. Peptides 1 and 2 were pentapeptides, whereas peptide 3 was a hexapeptide with a hydrophobic FMOC tail. Prior to investigating their selfassembly behaviors, a homogeneous solution of peptides 1 and 2 was prepared with a concentration of 5 mg/mL at a pH of 11. Through the addition of concentrated HCl, the self-assembly of the peptides can induce precipitation once the solution pH was lower than 5, and supramolecular hydrogels can be formed at a pH of 3.8 (Figure 1). The viscoelastic properties in Figure 2 indicated that the storage modulus (G′) was higher than the loss modulus (G′′), implying a gel characteristic rheological behavior of the solid-like materials. The interior morphology of the supramolecular hydrogels was examined by transmission electron microscopy (TEM). The corresponding results presented in Figure 1 indicated the formation of long nanofibers with the width of around 30-50 nm in the supramolecular hydrogels. However, when introducing concentrated NaOH to a solution of peptide 3 at a low pH of 4, there was no supramolecular hydrogel formation within a pH range from 4 to 11 even when the concentration of the peptide reached 15 mg/mL. Only a small amount of precipitation was presented when the solution pH was higher than a value of 8. This interesting result strongly suggested that the balance between hydrophobicity and hydrophilicity was extremely important for a molecular hydrogelator.31 To understand the molecular arrangement in the nanofibers of the supramolecular hydrogels, circular dichroism (CD, Figure

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Figure 3. CD (a) and FT-IR (b) spectra of the supramolecular hydrogels self-assembled from peptides 1 (Gel1) and 2 (Gel2) at a pH of 3.8.

Figure 4. XRD spectra of the supramolecular hydrogels self-assembled from peptides 1 (Gel1, a) and 2 (Gel2, b) at a pH of 3.8.

3a), FT-IR (Figure 3b), and WXRD (Figure 4) measurements were performed. With respect to the supramolecular hydrogel self-assembled from peptide 1 (Gel1), the negative band centered at ∼219 nm was attributed to the peptide n-π* transition.24,32 This CD signal shared a common feature with that of β-sheet conformation of a polypeptide,33 suggesting that the selfassembly of peptide 1 led to a β-sheet like superstructure. Besides, the negative broad band near 245-285 nm could also be observed in the CD spectrum of Gel1, and it was mainly due to the π-π* transition of terminal aromatic moiety.22,24,33 From the FT-IR spectrum of Gel1, the absorbance maxima of amide I frequency was at ∼1633 cm-1, a typical band of β-sheet like superstructure formed via intermolecular hydrogen bonding interactions.27,28 And the relatively weak peak at 1689 cm-1 was indicative of antiparallel alignment of the intermolecular hydrogen bonds among peptide backbones.28 From the WXRD data in Figure 4, Gel1 had a diffraction peak at ∼19.5° corresponding to a spacing of d ) 4.63 Å (d ) nλ/sin 2θ), also implying that self-assembly of peptide 1 induced the formation of a β-sheet like superstructure.34 Here, the diffraction peak at ∼40° (d ) 2.4 Å) mainly corresponded to the size of the peptide 1 (calculated as d ) 2.74 Å through Chem3D simulation). The CD, FT-IR, and WXRD spectra of the supramolecular hydrogel self-assembled from peptide 2 (Gel2) were similar with these of Gel1. The negative band near 219 nm (peptide n-π* transition) in the CD spectrum implied a β-sheet like superstructure resulted from the self-assembly of the peptide 2. Also, the negative band centered at ∼276 nm was mainly attributed

Figure 5. Emission spectra of the solution and supramolecular hydrogels of peptides 1 and 2.

to the π-π* transition of terminal aromatic moiety. The strong peak at ∼1634 cm-1 and relatively weak peak at ∼1689 cm-1 in the FT-IR spectrum suggested the β-sheet like superstructure formed via antiparallel hydrogen bonding interactions of the peptide backbones.27,28 Moreover, the signal at ∼20° (d ) 4.51 Å) in the WXRD spectrum also corresponded to the diffraction of the β-sheet like superstructure in the nanofibers. The data of CD, FT-IR, and WXRD spectra provided the evidence to demonstrate that the self-assembly of peptides 1 and 2 resulted in the formation of β-sheet like superstructure in the nanofibers through the hydrogen bonding interactions of the peptide backbones. As a hydrophobic tail, the detailed environment of the fluorenyl ring in the nanofibers was also examined, and the emission spectra are displayed in Figure 5. The solution of peptides 1 and 2 had an announced emission peak at ∼320 nm, which shifted to ∼332 nm after the pH triggered self-assembly. This red shift indicated that two fluorenyl rings overlapped through an antiparallel fashion in the nanofibers.22,23 Moreover, a relatively weak peak centered at ∼460 nm appeared in the emission spectra of the supramolecular hydrogels, suggesting that more than two of a few fluorenyl rings stacked efficiently in the hydrogels via π-π interactions, similar to the case of π-stacked polyfluorenes.35,36 In the current systems, increasing the pH value with NaOH to a neutral one can induce complete dissolution of the supramolecular hydrogels. This reversible self-assembly upon pH alternation has been also reported in previous studies, and the proposed reason was attributed to the intermolecular electrostatic repulsion.26-28,37 To determine whether this inter-

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Figure 6. Acid-base titration curves of peptides 1, 2, and 3 at a concentration of 2 mg/mL.

Figure 7. Optical and TEM images of the supramolecular hydrogel resulting from the coassembly of oppositely charged peptides 1 and 3 (Gel13) as well as the hydrogel resulting from the coassembly of oppositely charged peptides 2 and 3 (Gel23) at a neutral pH.

molecular electrostatic repulsion resulted in the failure of the self-assembly of the peptides, the pKa’s of peptides 1, 2, and 3 were examined via the classic acid-base titration. All the titrations started at a pH where the molecules have already been in their aggregated state to avoid kinetic effects of the selfassembly. As shown in Figure 6, the titrations of peptides 1 and 2 presented sharp transitions (Figure 6a and b), corresponding to two apparent pKa’s, mainly corresponding to the deprotonation of the more solvent accessible terminal carboxylic acid groups of peptides 1 and 2. However, it was difficult to exactly determine the pKa’s of Asp and Arg residues in the peptide backbones, implying that their protonation/deprotonation processes occurred slowly, with variations of acidity due to the local microenvironments within the nanofibers.28 It was clear from these results that aggregation changed the apparent pKa’s of the acid and amine groups in the peptide backbones, which was consistent with recent reports in the literature.26,38 The titration of peptide 3 presented a similar profile as peptides 1 and 2. The sharp transition mainly corresponded to the pKa of terminal carboxylic acid, and the slight transition possibly originated from one or more amines of Lys residues in the

peptide backbone. From the titration results of the peptides 1 and 2, at a neutral pH, either peptide 1 or 2 was ionized and loaded negative charges, which maintained the peptide molecules solubilized through the electrostatic repulsion from other negatively charged peptide molecules despite the existence of hydrophobic FMOC tails. Here, combining the information from CD, FT-IR, WXRD, emission spectra, and acid-base titrations, the mechanism for the pH triggered self-assembly of peptides 1 and 2 could be listed as follows. At a neutral pH or higher one, the intermolecular electrostatic repulsion resulted in the dissolution of the peptide molecules. However, through the acidifying treatment for deprotonation, the peptide backbones can carry out β-sheet like arrangement via antiparallel hydrogen bonding interactions, and the fluorenyl rings connected to the peptide backbones as hydrophobic tails were thus positioned to π-stack with each other in antiparallel fashion, resulting in the formation of nanofibers. Subsequently, these nanofibers entangled with each other and trapped solvent via surface tension to form supramolecular hydrogels. Coassembly of Oppositely Charged Peptides. As described above, the self-assembly of peptides 1 and 2 can not be ac-

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Figure 8. Oscillatory rheology of the supramolecular hydrogel resulting from the coassembly of oppositely charged peptides 1 and 3 (Gel13) as well as the hydrogel resulting from the coassembly of oppositely charged peptides 2 and 3 (Gel23) at a neutral pH.

complished at a neutral pH or higher one due to the intermolecular electrostatic repulsion. If another peptide loading opposite charges was introduced to this system, the initial intermolecular electrostatic repulsion can be expected to translate to electrostatic attraction, which may provide a potential chance for the coassembly of the oppositely charged peptides. From the titration curves revealed in Figure 6, peptides 1 and 2 were negatively charged at a neutral pH, whereas peptide 3 loaded positive charges. When mixing an equal amount of peptide 1 or 2 with peptide 3 in distilled water at a neutral pH, a supramolecular hydrogel can be formed within 12 h (Figure 7). On the basis of our experiments, it was found that these supramolecular hydrogels can only be obtained when the total concentration of peptide 1 and 3 or 2 and 3 reached 11 or 20 mg/mL. Moreover, with the same total concentration of the oppositely charged peptides, the supramolecular hydrogels can also be formed when slightly altering the composition of the oppositely charged peptides. However, as a control, the same concentration of peptide 1 (11 mg/mL) or 2 (20 mg/mL) can not self-assemble into a supramolecular hydrogel at a neutral pH within 2 weeks. All these results were distinct with the previous reports that the coassembly of oppositely charged peptides can be accomplished rapidly at a low concentration.27-30 The main reason was the different flexibility of peptide chains. In these reported systems, the peptides had no29,30 or flexible hydrophobic tails.27,28 Besides, the peptide backbones are generally composed of at

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Figure 10. XRD spectra of the supramolecular hydrogel resulting from the coassembly of oppositely charged peptides 1 and 3 (Gel13, a) as well as the hydrogel resulting from the coassembly of oppositely charged peptides 2 and 3 (Gel23, b) at a neutral pH.

least ten amino acid residues. However, as presented in Scheme 1, the backbones of the peptides prepared in this study were short, and there were rigid fluorenyl rings connected to the peptide backbones as hydrophobic tails. Consequently, the flexibility of these peptide chains was relatively weaker. Besides, the rigid fluorenyl rings can induce steric hindrance when the oppositely charged peptides began to be in contact with each other even though these fluorenyl rings can provide π-stacking in antiparallel fashion for the formation of nanofibers in the following discussion. All these factors were not in favor of the appearance of electrostatic attraction of the oppositely charged peptides. As a result, a relatively slow coassembly of the oppositely charged peptides was presented, and a higher concentration of the oppositely charged peptides was necessary for the supramolecular hydrogel formation. Anyway, no hydrogel formation in the control experiment strongly demonstrated that the electrostatic attraction triggered the coassembly of the oppositely charged peptides at a neutral pH. The supramolecular hydrogels resulting from the coassembly of the oppositely charged peptides were characterized by TEM, oscillatory rheology, CD, FT-IR, WXRD, and fluorescence spectroscopy. Figure 7 displays the TEM images of the supramolecular hydrogels. It can be found that long nanofibers with the width of around 40-60 nm exist in the hydrogels. The viscoelastic properties of the supramolecular hydrogels are

Figure 9. CD (a) and FT-IR (b) spectra of the supramolecular hydrogel resulting from the coassembly of oppositely charged peptides 1 and 3 (Gel13) as well as the hydrogel resulting from the coassembly of oppositely charged peptides 2 and 3 (Gel23) at a neutral pH.

Coassembly of Oppositely Charged Short Peptides

Figure 11. Emission spectra of the supramolecular hydrogel resulted from the coassembly of oppositely charged peptides 1 and 3 (Gel13) as well as the hydrogel resulted from the coassembly of oppositely charged peptides 2 and 3 (Gel23) at a neutral pH.

presented in Figure 8. The storage modulus (G′) was found to be approximately an order of magnitude larger than the loss modulus (G′′), indicative of an elastic rather than viscous material, and due to the higher entanglement of the nanofibers revealed in Figure 7, the storage modulus (G′) of the supramolecular hydrogels resulting from the coassembly of the oppositely charged peptides was larger than that of the hydrogels self-assembled from the individual peptide 1 or 2. Since TEM images demonstrated the formation of nanofibers in the supramolecular hydrogels, CD, FT-IR, WXRD, and fluorescence spectroscopy were employed to understand the arrangement of

J. Phys. Chem. B, Vol. 114, No. 7, 2010 2371 the oppositely charged peptide molecules in the nanofibers. As presented in Figure 9a, the negative band near 219 nm (peptide n-π* transition) in the CD spectra implies the presence of β-sheet like superstructure in the nanofibers of the supramolecular hydrogels. From the FT-IR spectra revealed in Figure 9b, the absorbance maxima of amide I frequency at ∼1635 cm-1 indicated the β-sheet like superstructure in the nanofibers, and this superstructure was formed via antiparallel hydrogen bonding interactions of the peptide backbones because of the weak band at ∼1685 cm-1. The diffraction peak at ∼19.6° (d ) 4.63 Å) in the WXRD spectra displayed in Figure 10 also demonstrated that coassembly of the oppositely charged peptides resulted in the formation of a β-sheet like superstructure. It should be noted that the signal at ∼28° (d ) 3.28 Å) mainly corresponded to the diffraction of the peptide 3 (calculated as d ) 3.26 Å through Chem3D simulation). The detailed environment of the fluorenyl rings in the nanofibers can be collected from the emission spectra of the supramolecular hydrogels. As shown in Figure 11, the supramolecular hydrogels had an emission peak at ∼340 nm, which presented ∼20 nm red shift in comparison with the emission spectrum of the solution of the individual peptides (Figure 5), implying that two fluorenyl rings overlapped in an antiparallel fashion. Similarly with the emission spectra of the supramolecular hydrogels self-assembled from the individual peptides, the broad peak centered at ∼460 nm with weak intensity implied that a small amount of multiple fluorenyl rings stacked with each other and moved less freely in the supramo-

SCHEME 2: Schematic Illustration of the Coassembly of the Oppositely Charged Peptides at a Neutral pHa

a

The electrostatic attraction provides a chance for the generation of π-stacking of fluorenyl groups and hydrogen bonding interactions of the peptide backbones, resulting in the formation of nanofibers with β-sheet like superstructure.

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lecular hydrogels resulting from the coassembly of the oppositely charged peptides. The spectroscopic techniques seemed to imply that the π-stacking of fluorenyl rings and the hydrogen bonding interactions of the peptide backbones resulted in the coassembly of the oppositely charged peptides. However, the self-assembly only occurred when mixing the oppositely charged peptides at a neutral pH where the individual peptide molecules were soluble, strongly suggesting that such a self-assembly was driven by an electrostatic attraction involving both positively and negatively charged peptide molecules and not the simple hydrophobic collapse involving one type or two different types of peptides. In other words, the electrostatic attraction made the oppositely charged peptides contact with each other, providing a chance for the β-sheet like arrangement of the peptide backbones through hydrogen bonding interactions, and the fluorenyl rings connected to the peptide backbones as hydrophobic tails were thus positioned to π-stack with each other in antiparallel fashion, resulting in the formation of nanofibers and ultimate supramolecular hydrogels (Scheme 2). Therefore, the oppositely charged peptides should be thoroughly mixed together within the formed nanofibers since the electrostatic attraction among them triggered the self-assembly. Conclusions In summary, we demonstrated a strategy to form supramolecular hydrogels triggered by electrostatic attraction of oppositely charged peptides with rigid FMOC tails. The TEM observations exhibited a fibroid morphology in these supramolecular hydrogels. The spectroscopic techniques indicated that the hydrogen bonding interactions induced a β-sheet like arrangement of the peptide backbones, and the fluorenyl rings connected to the peptide backbones as hydrophobic tails were thus positioned to π-stack with each other in antiparallel fashion, resulting in the formation of nanofibers and ultimate supramolecular hydrogels. Due to the weak flexibility of peptide chains and steric hindrance of rigid fluorenyl rings during the initial process of the coassembly of the oppositely charged peptides, a relatively slow self-assembly was presented, and a higher concentration of the mixed peptides was necessary for the supramolecular hydrogel formation. Acknowledgment. We acknowledge the financial support from the National Natural Science Foundation of China (20774069, 20974083) and National Key Basic Research Program of China (2005CB623903, 2009CB930300). References and Notes (1) Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312.

Xu et al. (2) MacDonald, J. C.; Whitesides, G. M. Chem. ReV. 1994, 94, 2383. (3) Zimmerman, S. C.; Zeng, F.; Reichert, D. E. C.; Kolotuchin, S. V. Science 1996, 271, 1095. (4) Trrech, P.; Weiss, R. G. Chem. ReV. 1997, 97, 3133. (5) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869. (6) Estroff, L. A.; Hamilton, A. D. Chem. ReV. 2004, 104, 1201. (7) Flory, P. J. Faraday Discuss 1974, 57, 7. (8) Keller, A. Faraday Discuss 1995, 101, 1. (9) Wang, R.; Geiger, C.; Chen, L.; Swanson, B.; Whitten, D. G. J. Am. Chem. Soc. 2000, 122, 2399. (10) Sakurai, K.; Jeong, Y.; Koumoto, K.; Friggeri, A.; Gronwald, O.; Sakurai, K.; Okamoto, S.; Inoue, K.; Shinkai, S. Langmuir 2003, 19, 8211. (11) Langer, R.; Vacanti, J. P. Science 1993, 260, 920. (12) Wang, Y.; Ameer, G. A.; Sheppard, B. J.; Langer, R. Nat. Biotechnol. 2002, 20, 602. (13) Kretsinger, J. K.; Haines, L. A.; Ozbas, B.; Pochan, D. J.; Schneider, J. P. Biomaterials 2005, 26, 5177. (14) Zhou, M.; Smith, A. M.; Das, A. K.; Hodson, N. W.; Collins, R. F.; Ulijn, R. V.; GoughJ., E. Biomaterials 2009, 30, 2523. (15) Smith, A. M.; Acquah, S. F. A.; Bone, N.; Kroto, H. W.; Ryadnov, M. G.; Stevens, M. S. P.; Walton, D. R. M.; Woolfson, D. N. Angew. Chem., Int. Ed. 2005, 44, 325. (16) Measey, T. J.; Schweitzer-Stenner, R. J. Am. Chem. Soc. 2006, 128, 13324. (17) Haines, L. A.; Rajagopal, K.; Ozbas, B.; Salick, D. A.; Pochan, D. J.; Schneider, J. P. J. Am. Chem. Soc. 2005, 127, 17025. (18) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2003, 303, 1352. (19) Reches, M.; Gazit, E. Science 2003, 300, 625. (20) Reches, M.; Gazit, E. Nano Lett. 2004, 4, 581. (21) Reches, M.; Gazit, E. Isr. J. Chem. 2005, 45, 363. (22) Zhang, Y.; Gu, H. W.; Yang, Z. M.; Xu, B. J. Am. Chem. Soc. 2003, 125, 13680. (23) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. F.; Saiani, A.; Gough, J. E.; Ulijn, R. V. AdV. Mater. 2006, 18, 611. (24) Yang, Z. M.; Liang, G. L.; Wang, L.; Xu, B. J. Am. Chem. Soc. 2006, 128, 3038. (25) Yang, Z. M.; Liang, G. L.; Ma, M. L.; Gao, Y.; Xu, B. J. Mater. Chem. 2007, 17, 850. (26) Tang, C.; Smith, A. M.; Collins, R. F.; Ulijn, R. V.; Saiani, A. Langmuir 2009, 25, 9447. (27) Niece, K. L.; Hartgerink, J. D.; Donners, J. J. J. M.; Stupp, S. I. J. Am. Chem. Soc. 2003, 125, 7146. (28) Behanna, H. A.; Donners, J. J. J. M.; Gordon, A. C.; Stupp, S. I. J. Am. Chem. Soc. 2005, 127, 1193. (29) Ramachandran, S.; Tseng, Y.; Yu, Y. B. Biomacromolecules 2005, 6, 1316. (30) Ramachandran, S.; Flynn, P.; Tseng, Y.; Yu, Y. B. Chem. Mater. 2005, 17, 6583. (31) Yang, Z. M.; Liang, G. L.; Xu, B. Chem. Commun. 2006, 738. (32) Yang, Z. M.; Liang, G. L.; Ma, M. L.; Abbah, A. S.; Lu, W. W.; Xu, B. Chem. Commun. 2007, 843. (33) Berova, N.; Nakanishi, K.; Woody, R. W., Eds. Circular Dichroism: Principles and Applications, 2nd ed.; Wiley-VCH: New York, 2000. (34) Hamley, I. W. Angew. Chem., Int. Ed. 2007, 46, 8128. (35) Yang, Z. M.; Xu, K. M.; Wang, L.; Gu, H. W.; Wei, H.; Zhang, M. J.; Xu, B. Chem. Commun. 2005, 4414. (36) Rathore, R.; Abdelwahed, S. H.; Guzei, I. A. J. Am. Chem. Soc. 2003, 125, 8712. (37) Jin, Y.; Xu, X. D.; Chen, C. S.; Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Macromol. Rapid Commun. 2008, 29, 1726. (38) Ohaski, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem. 2002, 13, 510.

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