Morphology Transformation via pH-Triggered Self-Assembly of

Dec 5, 2011 - Afzal Shah , Maria Saleem Malik , Gul Shahzada Khan , Erum Nosheen , Faiza Jan Iftikhar , Farman Ali Khan , Shyam S. Shukla , Mohamed ...
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Morphology Transformation via pH-Triggered Self-Assembly of Peptides Si-Yong Qin, Sheng-Sheng Xu, Ren-Xi Zhuo, and Xian-Zheng Zhang* Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: Three flexible peptides (P1: (C17H35CO-NHGRGDG)2KG; P2: (Fmoc-GRGDG)2KG; P3: (CH3CO-NHGRGDG)2KG) self-assembled to form a variety of morphologically distinct assemblies at different pHs. P1 formed nanofibers at pH 3, then self-assembled into nanospheres with pH up to 6 and further changed to lamellar structures when the pH value was further increased to 10. P2 aggregated into an entwined network structure at pH 3, and then selfassembled into well-defined nanospheres, lamellar structures, and vesicles via adjusting the pH value. However, P3 did not self-assemble into well-ordered nanostructures, presumably due to the absence of a large hydrophobic group. The varying self-assembly behaviors of the peptides at different pHs are attributed to molecular conformational changes. These self-assembled supramolecular materials might contribute to the development of new peptide-based biomaterials.

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INTRODUCTION Self-assembly based on biological building blocks such as peptides and proteins has attracted increasing interest due to the biodegradability, biocompatibility, and a wide range of applications in biomedicine and nanotechnology for these supramolecular materials.1 The self-assembly of peptides is important in life processes, especially to the pathogenesis of certain neurodegenerative diseases.2−6 Driven by cooperative noncovalent interactions such as hydrogen bonding, hydrophobic forces, electrostatic interactions, π−π stacking, and nonspecific van der Waals forces, peptides can spontaneously form well-defined nanostructures.7 Self-assembled peptides exhibit a range of structural architectures including nanorods,8 -tubes,9,10 -fibers,11,12 -spheres,13,14 and -donuts15 as well as conventional micelles.16 These nanostructures have been widely used as structural scaffoldings, selective transport channels, and templates for mineral formation.17 Zhang et al. reported control over peptide self-assembly by tuning the amphiphilicity of the building blocks, which led to the conversion between well-defined nanostructures and random aggregates.18−20 With respect to specific applications such as controlled drug delivery or models of pathogenic peptide assemblies, a single architecture may not provide the flexibility that some biological systems require. Thus, controlling the peptide self-assembly behavior is significant. In recent years, chemists have successfully constructed different nanoarchitectures that are sensitive to external stimuli. Then, different well-ordered morphological transformations can be triggered by changing the self-assembly time,21 adding new reagents,22 exposing to environmental stimuli,23−25 or judicious modification of the molecular structures.26 The transformations © 2011 American Chemical Society

of the self-assemblies are ascribed to the alteration of predominant weak interactions during the self-assembly. Among the weak forces, hydrophobic interactions and hydrogen bonding are ubiquitous in biological systems, and altering the balance between these forces may further affect other noncovalent interactions significantly, which thereafter tunes the self-assembly behavior and corresponding morphology. Better understanding of how noncovalent interactions induce morphological changes in a controlled manner is still a critical challenge in the field of biological self-assembly. Here, we aimed to investigate the morphological transformation of self-assembled peptides via rationally designing the peptide structure. Recently, U-shaped molecules, consisting of a linker and two arms, were reported to form vesicles via bilayer self-assembly.23,27,28 We assumed that if the arms of the U-shaped molecule were expanded from two ends to form a linear molecule or staggered from each other to form a Z-shaped molecule, the self-assembled morphology may change accordingly. Keeping this in mind, we developed a set of flexible molecules that would exhibit reversible structural transformation between the U-shaped and Z-shaped or even linear type as illustrated in Figure 1, providing the possibility to accomplish a conversion in morphology. The RGD containing peptide sequence was introduced to act as the hydrophilic backbone and to provide pH sensitivity due to the basic arginine and acidic aspartic acid residues. A pH change might trigger the Special Issue: Bioinspired Assemblies and Interfaces Received: July 27, 2011 Revised: November 30, 2011 Published: December 5, 2011 2083

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activating the stearic acid by HBTU and DIEA in a DMF/ dichloromethane (DCM) mixed solution at the end of the synthesis. Then, the resin was respectively washed with DMF, MeOH, and DCM three times and dried under vacuum overnight. Cleavage of the expected peptide and removal of side chain protected groups from the resin were performed using a cocktail of TFA, phenol, EDT, thioanisole, and deionized (DI) water, in the volume ratio of 82.5:7.5:2.5:5:2.5. After stirring for 2 h at room temperature, the cleavage mixture and subsequent TFA washing solution were combined. The collected solution was concentrated to a viscous solution by rotary evaporation and then dropped in 10-fold cold ether to precipitate the product. Further washing with cold ether three times was employed to remove TFA residual and impurities. The precipitate was quickly centrifugated and dried under vacuum for 24 h. ESI-MS, P1: calculated 1620.0, [M+H]+ at m/z 1621.1 and [M+2H]2+ at m/z 811.5 were observed; P2: calculated 1532.6, [M+H]+ at m/z 1533.6 and [M+2H]2+ at m/z 767.3 were observed; P3: calculated 1171.5, [M+H]+ at m/z 1172.6 and [M+2H]2+ at m/z 587.1 were observed. (Figure S1−3 in the Supporting Information). Acid−Base Titration. The solution of peptides (P1 and P2) was prepared in distilled water at a concentration of 0.5 mg/mL and 1 mg/mL, respectively. For the pKa titrations of basic peptides 1 and 2, 0.01 M HCl aqueous solution was added in 10−50 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. Transmission Electron Microscopy (TEM). The microstructures of self-assemblies were observed by TEM (JEM-2010, Japan) with an accelerating voltage of 100 kV. After the preparation, the solutions of different pHs were kept at room temperature for one night to obtain stable self-assemblies. The samples were prepared by dipping a copper grid with carbon film into the solution containing self-assemblies. After the deposition, the samples were dried in air for the observations. Circular Dichroism (CD). The self-assembled peptide solutions (2 mg/mL) 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. UV/Vis Absorption/Fluorescence Spectroscopy. Fluorescence spectra of the self-assembled P2 solutions at different pHs or concentrations were recorded on a LS55 luminescence spectrometry (Perkin-Elmer) with excitation at 265 nm, and emission data range between 300 and 600 nm. The UV/vis absorption of aromatic groups of P2 solutions was also measured by using a UV/vis spectrophotometer (Perkin-Elmer Lambda Bio 40 UV/vis spectrometer, USA) in the wavelength region from 225 to 400 nm. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectra of the self-assembled peptides were performed on an AVATAR 360 spectrometer. Prior to the measurements, the freeze-dried selfassembled peptides were pressed with potassium bromide (KBr) powder to form pellets.

Figure 1. The optimized molecular conformation and total energy of three conformational types of P1 via MM2 method. (a) Z-shaped conformation with total energy of −14.8 kJ·mol−1; (b) U-shaped conformation with total energy of 35.4 kJ·mol−1; and (c) linear style conformation with total energy of 336.9 kJ·mol−1.

protonation/deprotonation of the amino- and carboxyl- groups, and the change of static electric force would lead to a corresponding change in the molecular arrangement. In parallel, we sought to exploit the tendency of hydrophobicity to affect peptide self-assembly behavior.29 Hydrophobic groups were introduced into the arms of the peptides, and their influence on the self-assembly was investigated.

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EXPERIMENTAL SECTION

Materials. N-Fluorenyl-9-methoxycarbonyl (Fmoc) protected L-amino acids (Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Fmoc)-OH), 2-chlorotrityl chloride resin (2-CTC resin) (100−200 mesh, loading: 1.38 mmol/g), 1-hydroxybenzotriazole (HOBt), O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU), 1,2-ethylenedithiol (EDT), thioanisole, and piperidine were purchased from GL Biochem (Shanghai) Ltd. (China) and used as received. Trifluoroacetic acid (TFA), and stearic acid (C17H35COOH) were provided by Shanghai Reagent Chemical Co. (China). Diisopropylethylamine (DIEA), dimethylformamide (DMF) were distilled before used. All other reagents and solvents were used without further purification. Synthesis of Peptides (P1−3). The peptides (P1−3, P1: (C 17H 35CO-NH-GRGDG)2 KG; P2: (Fmoc-GRGDG)2 KG; P3: (CH3CO-NH-GRGDG)2KG) were synthesized manually on the 2-chlorotrityl chloride employing a standard Fmoc chemistry. Before the synthesis, the resin (1 g) was immersed in DMF for 0.5 h. Then Fmoc-Gly-OH was fixed on the swelling resin with a DMF solution of the mixture of Fmoc-Gly-OH (2 equiv relative to resin loading) and DIEA (6 equiv relative to Fmoc-Gly-OH) stirred for 2 h at room temperature. After removing the reaction solution, the resin was washed with DMF four times. Methanol was used to react with residual active chloride of the resin. Subsequently, 20% piperidine/ DMF (V/V) solution was introduced to the resin to remove the Fmoc groups from the amino acid twice. After stirring for 30 min (2 × 15 min) at room temperature, the reaction solution was drained off, and the resin was washed with DMF four times. The exposed amino resin was tested as a purple color by the Kaiser reagent and admitted to the next step. Hereafter, a DMF solution of the mixture of Fmoc protected amino acid (2 equiv), HBTU (2.4 equiv), HOBt (2.4 equiv), and DIEA (6 equiv) was added. After stirring for 2 h at room temperature, the reaction solution was drained off, and the resin was washed with DMF four times. The amino-protected resin was tracked by the Kaiser reagent to text the completion of reaction. After repetition of the deprotecting and acylating reactions for amino acids loading, the fatty tails were conjugated to the peptide segments after

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RESULTS AND DISCUSSION To achieve the transformation of the self-assembled morphology, we designed three flexible peptides (Figure 2), which were

Figure 2. Chemical structures of three structural complementary peptides (P1−P3).

expected to exhibit reversible structural transformations between Z-shaped and U-shaped or linear conformations. 2084

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at pH 10 (Figure 4d). However, P3 could not self-assemble into well-ordered nanostructures, presumably due to the absence of large hydrophobic groups. To explain morphological transformations in peptide assemblies, a hypothesis proposed in the literature focused on changes in the peptide secondary structures.30,31 In order to further understand the conformational properties of peptides P1 and P2, CD, FT-IR, and UV/vis absorption/fluorescence spectroscopy were used to study the molecular interactions involved in the self-assembly behavior. According to the literature,8,32,33 negative CD bands near 208 and 222 nm, and a positive band near 192 nm are characteristic of the α-helix conformation of peptides, while a negative broad band near 216 nm and positive band near 195 nm correspond to the β-sheet conformation. Figure 5a−c illustrates the CD spectra of P1 at various pH values. From Figure 5a, negative peaks appearing at around 225 nm, 206 and 195 nm and a positive peak crossing the x-axis near 190 nm of P1 of the self-assembled nanofibers (pH = 3) suggested an α-helix structure mixed with a partial random-coil conformation. By contrast, a β-sheet-like conformation could be found in the self-assembled nanospheres (pH = 6) of P1, based on the broad negative band near 216 nm (Figure 5b). In the self-assembled lamellar structures (pH = 10) P1 adopted an α-helix-like conformation containing partial β-sheet conformation, which was supported by the negative bands near 225 and 216 nm, together with the positive band at 192 nm (Figure 5c). To further certify the above verdict, FT-IR was employed to study the self-assembled secondary structure of the peptides through analyzing the absorption band of amide bond in the region from 1800 to 1400 cm−1. According to the literature,34 an α-helix exhibits absorption bands at 1650−1660 cm−1 and ∼1545 cm−1 for amide I and amide II bands, respectively, while the β-sheet conformation has the characteristic amide I and amide II absorption bands at 1620−1640 cm−1 and ∼1530 cm−1, respectively. For the nanofibers, two dominant peaks are located at around 1655 cm−1 and 1545 cm−1 (Figure 5d), suggesting that the main secondary structure was an α-helix, confirming the findings obtained by CD. The absorption bands at around 1638 cm−1 and 1530 cm−1 indicated the presence of a β-sheet-like structure in the nanospheres (Figure 5e). The selfassembled lamellar structures adopting an α-helix-like conformation containing partial β-sheet conformation was further supported by the absorption bands at 1645 cm−1, 1634 cm−1 and 1550 cm−1 in Figure 5f. As for P2, we selected the morphologies at pH 5 and 7. The negative band near 227 nm, together with the positive band at 192 nm indicated that the nanospheres formed at lower pH exhibited an α-helix-like structure mixed with partial random-coil conformation (Figure 6a). The higher pH lamellar structures exhibited negative bands near 202 and 218 nm, together with a positive band at 192 nm, indicative of a predominant randomcoil conformation with partial α-helix structure (Figure 6b). These findings were further supported by FT-IR spectra. The absorption bands at near 1660 cm−1 and 1540 cm−1 contributed to α-helix secondary structure (Figure 6c). In neutral conditions, the self-assembled lamellar structure of P2 exhibits broad absorption bands 1670 cm−1 and 1540 cm−1, consistent with a random-coil conformation mixed with partial α-helix structure (Figure 6d). The CD and FT-IR data indicated that the secondary structures of P1 and P2 did not exhibit regular transformation between the most common secondary structures such as

The RGD peptide sequence, which contains charged aminoand carboxyl- groups, was introduced to make up the peptide backbone and can exploited as a pH sensitive trigger for morphological changes. C17H35CO, was selected to act as the hydrophobic tails of P1 and perform as the arms governing the reversible transformations among the U, Z, and linear states. Fmoc groups were incorporated into P2 to investigate the influence on conversion between the rigid aromatic−aromatic π−π conjugated system and the flexible alkyl group. CH3CO was introduced to P3 as a less hydrophobic alternative to the other two peptides. According to TEM images, P1 formed nanofibers at pH 3 (Figure 3a), which transformed to nanospheres with pH up to

Figure 3. TEM images of self-assembled P1 at different pHs. P1 formed insoluble fibrillar structures at pH 3 (a), then transformed into nanospheres at pH 6 (d), and further tuned to soluble lamellar aggregates at pH 10 (f).

6 (Figure 3d), then further turned into lamellar structures when the pH was further increased to 10 (Figure 3f). P2 aggregated into an entwined network structure at pH 3 (Figure 4a), and

Figure 4. TEM images of self-assembled P2 in different pHs. P2 formed an entwined network structure at pH 3 (a), then selfassembled into nanospheres at pH 5 (b), and further tuned to soluble lamellar structure aggregates at pH 7 (c), and vesicles at pH 10 (d). Higher magnification TEM image of a vesicle (e).

then self-assembled into well-defined nanospheres at pH 5 (Figure 4b), lamellar structures at pH 7 (Figure 4c), and vesicles 2085

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Figure 5. CD spectra (a−c) of the self-assembled P1 solutions and FT-IR (d−f) of the freeze-dried self-assembled P1 nanostructures. (a) CD spectrum of the self-assembled P1 solution at pH 3 with 1 mg/mL; (b) pH 6 and (c) pH 10. (d-f) FT-IR spectra of the freeze-dried self-assembled P1 at the above pHs, respectively.

Figure 6. CD spectra (a,b) of the self-assembled P2 solutions, and FT-IR (c,d) of the freeze-dried self-assembled P2 nanostructures. (a) CD spectrum of the self-assembled P2 solution at pH 5 with 1 mg/mL, (b) pH 7. (c,d) FT-IR spectra of the freeze-dried self-assembled P2 at the above pHs, respectively.

35.4 kJ·mol−1, and the linear conformation was 336.9 kJ·mol−1. Thus, the optimal conformation was Z-shaped due to the much lower total energy. However, it may convert to another conformation once receiving extra energy, and therefore the self-assembled morphologies may change accordingly. The fluorescence emission and UV/vis absorption spectra were used to investigate the influence of aromatic−aromatic π−π stacking interaction on self-assembly of P2. In the solution phase, the fluorescence spectra of P2 at different pHs (Figure 7a) and concentrations (Figure 7b) were recorded. According to Figure 7a, the central peaks at 313 nm showed slight red-shifts to 315 nm as the pH value increased from 3 to 7, which exhibited the existence of weak aromatic π−π stacking interactions. Simultaneously, the new weak band near 470 nm

α-helices, β-sheets, and coiled coils when morphologies of self-assemblies transformed. These results suggest that the hypothesis that secondary structural changes cause the transformation of the self-assembled architectures of peptide materials may be not the best interpretation for our system. We presumed that the morphological transformation might be triggered by the changes in the molecular conformation. The MM2 (minimize energy function, Chem 3D) force field was used to calculate the minimized energy conformation. Three representative conformations (Z-shaped, U-shaped, and linear style) were proposed here, and the corresponding minimized energy conformation was also calculated (see Figure 1). It was found that the total energy of the Z-shaped conformation of P1 was −14.8 kJ·mol−1, the U-shaped conformation was 2086

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Figure 7. Fluorescence emission (λex = 265 nm) spectra (a,b) and ultraviolet absorption spectra (c,d) of the self-assembled P2 solution: (a,c) at different pHs but same concentration of 1.0 mg/mL, and (b,d) at different concentrations but the same pH of 7.

UV/vis absorption spectroscopy. Fluorescence emission is more sensitive than UV/vis absorption spectroscopy, and the subtle red-shift can not be displayed in the UV/vis absorption spectra. Here, the peptide molecules consisting of charged amino acids can respond to the pH-stimulus. The pH change will trigger the change of static electric forces, which indirectly affect the hydrogen bondings of amino acid residue, and therefore the architectures of the self-assemblies may change correspondingly. Thus, adjusting the pH value may induce the morphological change. Meanwhile, we changed the pH and adjusted it to its original value, and the morphologies correspondingly changed into the original nanostructures. The transition among the different nanostructures was reversible and repeatable without any obvious fatigue effects. Figure S4a−c and Figure S4d−f (Supporting Information) show the nanoarchitectures of P1 and P2 when the pHs returned to their original values, respectively. This reversible conversion of self-assemblies upon pH alternation was also reported in previous studies, and the proposed reason was ascribed to the molecular electrostatic interaction.25 To determine whether the molecular electrostatic interaction resulted in a change of the peptide molecular conformation, the pKa’s of P1 and P2 were examined via the classic acid−base titration.37 From Figure 8a, it was found that P1 presented two transitions at pH 5.35 and 8.45, corresponding to the deprotonation of the carboxylic acid group of side chain in Asp and the guanidino group in Arg residue, respectively. As for P2, a similar profile was found in Figure 8b. The two transitions at pH 5.93 and 8.57 also corresponded to the pKa of the side chains of Asp and Arg, respectively. According to the molecular structures, there are three carboxylic acid groups and two guanidino groups, which should thus give rise to five transitions. The obtained results indicated that their protonation/deprotonation processes occurred slowly, with the overlap of some transitions of the charged groups. The differences of pKa presented in P1 and P2 indicate that even the same amino acids located at the same position show different ionization due to different hydrophobic blocks. For P1, at pH below 5.35, the carboxyl group of aspartic acid did not ionize and formed intermolecular hydrogen bonds,

Figure 8. Acid base titration curves of P1 and P2. (a) The acid base titration curve of P1 at a concentration of 0.5 mg/mL and (b) the curve of P2 at 1 mg/mL.

further demonstrated the π−π stacking interactions of the Fmoc groups.35,36 Then, the peak returned to 313 nm along with the pH further increasing, indicating that π−π interaction only happened at about pH 7 or it was stronger than at the other pH values. In other words, the aromatic−aromatic stacking interaction of Fmoc groups mainly participated in the formation of lamellar aggregates, but the formation of other self-assemblies was more likely to be due to hydrophobic rather than π−π stacking interactions. Meanwhile, the subtle shifts also occurred at different concentrations under the pH = 7 (Figure 7b). The peak at 312 nm slipped to 315 nm as the solution concentration changed from 0.01 mg/mL up to 1.0 mg/mL. This finding also confirmed that Fmoc not only played the role of the hydrophobic segment but also participated in aromatic−aromatic π−π stacking interaction for the lamellar aggregates of P2. UV−vis absorption of aromatic groups was also used to check the π−π stacking interactions of the Fmoc group (Figure 7c,d). From Figure 7c, the maximum absorption peak of the UV spectrum shifted from 263 to 265 nm when the pH increased from 3 to 7, and the peak dropped to 255 nm at pH 10. The results coincided with the above fluorescence data. However, the peaks of Figure 7d did not change as the solution concentration increased from 0.1 mg/mL to 1.0 mg/mL. The finding may be due to the different sensitivity of the fluorescence emission and 2087

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Scheme 1. Proposed Self-Assembly Mechanisms of P1 in Different pHsa

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Left: the optimized molecular conformations in different pHs via MM2 method; Middle: the hierarchically self-assembled metastructures; Right: the microcosmic morphologies under TEM. Hydrogen bonds were omitted for clarity.

peptide molecules, the secondary structure reversed back to the α-helix-like conformation, containing partial β-sheet conformation. The partial β-sheet conformation may be attributable to incomplete structural transformation. The proposed transformation mechanisms of the self-assembled P1 are illustrated in Scheme 1. Compared with P1, P2 demonstrated some different self-assembled morphologies at different pHs. At pH 3, P2 exhibited an entwined network instead of a nanofiber as shown in Figure 4a. The lack of a well-defined fiber in acidic conditions could be due to the fact that Fmoc is a rigid group, and the steric effect of the aromatic rings prevents the Fmoc groups from participating in twining with other molecules. At pH 5−6, self-assembled nanospheres of P2 were mainly attributed to strong hydrophobic interactions as well as hydrogen bond interactions (Figure 4b). The formation mechanism is similar to that of nanospheres of P1 mentioned above. However, the conformation that P2 adopted to selfassemble into nanospheres was different from P1 presumably due to the different hydrophobic groups on the two peptides. The self-assembled P2 nanospheres adopted the α-helix-like structure mixed with partial random-coil conformation. In neutral solution, P2 was ionized with negative charges, which made the peptide soluble despite the existence of hydrophobic Fmoc tails. The ordered π−π stacking and hydrogen bond interactions made the peptide molecules adopt a well-ordered side-by-side arrangement, which led to the formation of soluble lamellar structures (Figure 4c). Nevertheless, some hydrophobic edges of the nanosheet would be exposed to water. Due to the hydrophobic interaction, the lamellar structure was metastable, and the edges could fuse with other hydrophobic surfaces upon pH increase. So the sheets gradually tuned to vesicles when the pH value exceeded 8.57 (the pKa of guanidino group of Arg residues) (Figure 4d). The formation of the vesicle was due to the overlap of the Fmoc groups, keeping the hydrophilic peptides surrounded by water. The

leading to the peptides aggregation and poor dissolution in acid solution. The insoluble peptide could not stretch, and the hydrophobic interaction of alkyl segments favored the formation of fiber combined with hydrogen bond interaction (Figure 3a) via α-helix structure mixed with partial random-coil conformation. With the increasing pH, the primitive reticular nanofibers fused and shrunk, accompanied by aggregation of the nanofibers (Figure 3b,c). When the pH exceeded the first sharp transition (pKa1 5.35), the carboxyl group of Asp should deprotonate, weakening the hydrogen bond interaction. At the same time, the hydrophobic alkyl tail cannot be stabilized in water, and the hydrophobic core of the unilaminar nanosphere formed, leaving the hydrophilic peptide oriented outward. Then, the peptide segment took the parallel β-sheet-like conformation to self-assemble, and the salt-bridge interaction between the deprotonated Asp and protonated Arg of adjacent unilaminar nanospheres and the intermolecular hydrogen bond interactions drove them to form the multilaminar solid nanospheres via hierarchical self-assembly at pH 6 (Figure 3d). Upon further pH increase, the carboxyl groups of Asp deprotonated completely, and the electrostatic repulsion compelled the peptide molecules to become soluble and separate from each other. The second slight transition at pH 8.45 originated from the guanidino group of the Arg residue in the peptide backbone. At pH of around 8.45, the guanidino groups deprotonated, which impaired the formation of the intermolecular salt-bridges between the Asp and Arg side chains, and reduced electrostatic repulsion interactions from the guanidino group of Arg residues. Due to the lack of the saltbridge interaction, the multilamellar nanospheres began to disassemble, and the decreased electrostatic repulsion made the edges of the nanospheres fuse together (Figure 3e). The hydrogen bonds of the peptides in the backbone and the hydrophobic interaction of alkyl groups drove the peptide molecule rearrangement via side-by-side to lamellar structure aggregates at pH 10 (Figure 3f). Due to the rearrangement of the 2088

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hollow feature of the vesicles is evidenced by Figure 4e. The proposed vesicle formation of the self-assembled P2 is illustrated in Scheme 2.

AUTHOR INFORMATION

Corresponding Author *Tel./Fax: 86-27-68754509. E-mail address: xz-zhang@whu. edu.cn.

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Scheme 2. (a) The Chemical Structure of P2; (b) The Optimized Molecular Conformation via MM2 at pH 10; (c) Proposed Vesicle Formation Mechanism of the SelfAssembled P2a

ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology of China (2011CB606202, 2009CB930300).

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Hydrogen bonds were omitted for clarity.

It should be pointed out that P3 exhibited no well-defined nanostructure at varying pHs. The reason was ascribed to the principle that amphiphilicity is one of the molecular bases for self-assembly.18 Namely, to fabricate the self-assembled molecule, the ratio of the hydrophobic segment to hydrophilic group should be considered. In comparison with P1 and P2, the methyl group in P3 performing the role of hydrophobic group was not sufficiently hydrophobic to induce the well-ordered aggregation.

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CONCLUSIONS In summary, three peptides were prepared by standard Fmoc chemistry. By adjusting the pH of the self-assembled peptide systems, a smooth and reversible morphological transformation was achieved. At pH 3, P1 formed reticular nanofibers. With the pH increasing to 6, the nanofibers fused and shrunk, and the aggregation of the hydrophobic groups gave rise to the nanosphere structure. Upon further pH increasing, the edges of nanospheres gradually fused and further self-assembled into lamellar structure at pH 10. Compared with P1, P2 could not form nanofibers at pH 3, but formed vesicles at pH 10, which was attributed to the fact that Fmoc and alkyl groups have different rigidity. In contrast, P3 could not self-assemble into well-defined nanostructures due to the absence of large hydrophobic groups. Such morphological transitions likely originated from the molecular conformational changes of the hydrophobic building blocks and the flexible peptide backbone. This study might help to develop new peptide-based biomaterials.

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ASSOCIATED CONTENT * Supporting Information ESI-MS of P1, P2, and P3 as well as the TEM images of the reversible conversion of self-assemblies upon pH alternation. This information is available free of charge via the Internet at http://pubs.acs.org/. S

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