Temperature-induced reversible self-assembly of diphenylalanine peptide and the structural transition from organogel to crystalline nanowires
© Huang et al.; licensee Springer. 2014
Received: 1 November 2014
Accepted: 26 November 2014
Published: 3 December 2014
Controlling the self-assembly of diphenylalanine peptide (FF) into various nanoarchitectures has received great amounts of attention in recent years. Here, we report the temperature-induced reversible self-assembly of diphenylalanine peptide to microtubes, nanowires, or organogel in different solvents. We also find that the organogel in isopropanol transforms into crystalline flakes or nanowires when the temperature increases. The reversible self-assembly in polar solvents may be mainly controlled by electronic and aromatic interactions between the FF molecules themselves, which is associated with the dissociation equilibrium and significantly influenced by temperature. We found that the organogel in the isopropanol solvent made a unique transition to crystalline structures, a process that is driven by temperature and may be kinetically controlled. During the heating-cooling process, FF preferentially self-assembles to metastable nanofibers and organogel. They further transform to thermodynamically stable crystal structures via molecular rearrangement after introducing an external energy, such as the increasing temperature used in this study. The strategy demonstrated in this study provides an efficient way to controllably fabricate smart, temperature-responsive peptide nanomaterials and enriches the understanding of the growth mechanism of diphenylalanine peptide nanostructures.
Supramolecular self-assembly, a ubiquitously spontaneous process in nature, plays an important role in building highly ordered and functional structures in biology. It also provides a powerful tool for creating supramolecular nanostructures for various applications[1–5]. A substantial amount of research has focused on this issue, and various artificial self-assembling systems have been developed for peptides[1–4], peptide amphiphiles[5–7], and aromatic small molecules[8–10]. In particular, peptide-based supramolecular nanostructures have received great attention in recent years due to their excellent biocompatibility and functional diversity. Therefore, many peptide-based building blocks, including aromatic dipeptides[1, 4, 11–14], surfactant-like peptides, amyloid peptide fragments[16, 17], and cyclic peptides, have been designed and developed for the construction of organized supramolecular nanostructures. Among these peptide-based self-assembling molecules, diphenylalanine peptide (l-Phe-l-Phe, FF), which is the core recognition motif of Alzheimer's Aβ peptides, is a fascinating unit that can self-assemble into diverse structures, such as microtubes, nanowires, and microcrystals. These assembled nanomaterials are extremely attractive as potential building blocks in the field of sensors, imaging, nanofabrication, and so on[1, 4, 18–21].
Controlling the self-assembly of small molecules into highly ordered architectures is an interesting topic in supramolecular chemistry. For the diphenylalanine peptide, the use of different organic solvents is employed to control the self-assembly behavior. For instance, diphenylalanine self-assembles to hollow tubular structures in aqueous or methanol solutions[22–26]. A structural transition from FF microtubes to highly uniform nanowires was demonstrated in our previous study by introducing acetonitrile as a cosolvent in water. Interestingly, diphenylalanine also self-assembles in toluene or chloroform and forms amorphous nanofibers (gel), which are significantly different from the crystalline nanowires previously mentioned. Another control strategy is the addition of a surface in the assembly solution, such as glass[27, 28], or porous membrane. The coexistence of the solvent and the surface makes controlling the morphologies of the peptide assemblies easier and more efficient. In these studies, the self-assembly of diphenylalanine peptides generally occurred at room temperature. Previously, Heredia et al. demonstrated an irreversible phase transition of FF nanotubes from the hexagonal phase to another (most likely orthorhombic) crystalline phase at approximately 140°C to 150°C, indicating that temperature may be an important factor for controlling the structures of FF assemblies; however, the effect of temperature on the self-assembly of FF is often neglected. This study aims to develop a temperature-based control method for the self-assembly of diphenylalanine.
Temperature was considered an important parameter in the vapor-assisted self-assembly of diphenylalanine, in which a high temperature (e.g.,150°C, 220°C) was required for the structural transition or evaporation of FF molecules. For example, Ryu and Park reported a high temperature (150°C) aniline vapor aging process to synthesize a well-aligned peptide nanowire array starting from an amorphous peptide thin film. By increasing the temperature to 250°C, the FF molecules evaporated and self-assembled on a substrate to form single crystalline nanowires. These previous studies focus on the vapor-assisted self-assembly at high temperature; however, it is still unclear how the temperature (especially, temperatures lower than 100°C) influences the self-assembly of diphenylalanine in solvents or the structures of assemblies.
Herein, we report the temperature-induced reversible self-assembly of diphenylalanine peptide in solvents. Different solvents, including acetonitrile, acetonitrile-H2O, H2O, HFIP-H2O, and isopropanol, were chosen to demonstrate the assembly behavior. A temperature-responsive FF organogel in isopropanol is outlined. In this system, we further demonstrate a temperature-induced structural transition from an organogel to a crystalline flake-like structure in isopropanol. Additionally, the nanofibers in the organogel transferred into uniform crystalline nanowires with the assistance of a glass surface at an increased temperature. This temperature-based control process and the results of this study provide an efficient method for controlling the self-assembly of diphenylalanine peptide.
The lyophilized diphenylalanine peptide (NH2-Phe-Phe-COOH, FF) was purchased from Bachem (Bubendorf, Switzerland). The 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was bought from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and isopropanol were obtained from Aladdin Industrial Co. (Shanghai, China). All other reagents were of the highest grades available commercially.
Temperature-induced self-assembly of FF in different solvents
Suspensions were prepared using 2 mg diphenylalanine peptide added to 1 mL each of acetonitrile, acetonitrile-water (95:5 v/v), pure water (H2O), or isopropanol. The resulting suspensions were then heated to 90°C for 5 min, forming a transparent solution. The FF nanowires, microtubes, or transparent gels were formed during cooling to ambient temperature (25°C) without any disturbance.
Temperature-induced reversible self-assembly
After the formation of FF assemblies following the procedure mentioned above, the solution or gel containing FF assemblies was heated to 90°C for 5 min again. The assemblies were disassembled leading to the formation of a transparent solution. Then, the solution was cooled to 25°C without any disturbance. The disassembled FF molecules further self-assembled to nanowires, microtubes, or an organogel in different solvents again. All the heating-cooling experiments were carried out in the sealed tubes to prevent the volatilization of solvents. Three heating-cooling cycles were operated to confirm this reversible self-assembly process.
Temperature-induced structural transition in isopropanol
The FF organogel was stored at either -25°C for over 2 months or 25°C for 3 days. Next, 50 μL FF gel was deposited onto a microscopic glass coverslip and sequentially air-dried at -25°C and 25°C, respectively. The resulting samples were then observed by scanning electron microscopy.
Scanning electron microscopy
All the samples were sputter coated with platinum using an E1045 Pt-coater (Hitachi, Tokyo, Japan) and then imaged by using an S-4800 field emission scanning electron microscope (Hitachi High-Technologies Co., Tokyo, Japan) at an acceleration voltage of 5 kV. To observe the real structure of the organogel, the sample was prepared by flash freezing with liquid nitrogen followed by air drying at -45°C.
X-ray diffraction measurements were performed on a D8 Focus powder diffractometer (Bruker, Karlsruhe, Germany). The diffraction intensity of CuKa radiation (wavelength of 1.5418 nm) was measured under 40 kV and 40 mA with a scan rate of 2°/min in a 2θ range between 3° and 50°.
Size distribution measurement
The size distribution and mean diameter, d, of FF assemblies in HFIP-water were determined using a Zetasizer Nano (0.4 nm to 10 μm, Malvern Instruments, Worcestershire, UK) particle size analyzer. The FF self-assembly was performed in a quartz cell directly following the heating-cooling procedure. The initial temperature was set at 90°C, and then, the samples were cooled to 80°C, 70°C, 60°C, 50°C, 40°C, and 30°C. All the temperature points were maintained for 30 min to ensure the complete self-assembly of the FF. The experimental analysis was repeated three times.
Results and discussion
Interestingly, the formed nanowires or microtubes further disassembled when they were reheated to 90°C, again forming a transparent solution. During the cooling process (from 90°C to 25°C), the white FF assemblies were formed again in the solutions (Figure 2a,b,d,e and Additional file1: Figure S1a-b). More than three heating-cooling cycles were performed to confirm the reversible self-assembly phenomenon. SEM was used to observe the resulting samples after three cycles (Additional file1: Figure S2). The results indicated that the nanowires from both the first and third cycles had the same morphology and similar sizes. X-ray diffraction (XRD) was used to characterize the crystal structure using the FF nanowires formed in acetonitrile-H2O (third cycle) solution, a commonly used self-assembly medium. As shown in Additional file1: Figure S3, the XRD spectrum was identical to that in our previous study, in which a hexagonal crystal structure was observed.
In addition to the temperature-induced self-assembly, HFIP was often used in previous studies to dissolve diphenylalanine peptide and then initiate the supramolecular self-assembly in aqueous or organic solvents. To verify that the reversible self-assembly behavior of FF was dependent on temperature alone, we prepared nanowires using the HFIP-initiated self-assembly method (see Additional file1). We found that the formed FF nanowires could also again disassemble at high temperature (90°C) and self-assemble again during the cooling process (Additional file1: Figure S1d-f). These results indicate that temperature plays a definite role in the self-assembly and disassembly of the FF peptide. Furthermore, the FF molecules self-assembled into nanostructures in the solution at low temperature (e.g., 25°C), and the resulting nanostructures then disassembled at high temperature (e.g., 90°C) again. This temperature-induced self-assembly and disassembly is perfectly reversible in different solvents.
As we know, amino acids and some peptides exist as zwitterions in aqueous solutions. The carboxylate and amino groups can be ionized as the deprotonated (-COO-) and protonated (-NH3+) forms. Due to the rapid self-assembly of FF in solvents, it is difficult to determine the pKa values. Previously, temperature has been shown to play an important role in the dissociation of -NH3+, while having less influence on -COOH[32, 33]. By increasing temperature from 25°C to 90°C, the value of pK2 (-logK a, -NH3+) decreased, leading to a significant shift of the equilibrium to the -NH2 side at a fixed pH value (e.g., pH ~ 7 in our case). As a result, this dissociation equilibrium led to the change in electronic interactions and hydrogen bonding between FF-FF and FF-solvent molecules. In this case, a high solubility was achieved at 90°C. With decreasing temperature, the dissociation equilibrium shifts to the -NH3+ side. In this case, the electronic repulsion was eliminated, and the strength of hydrogen bonds (e.g., FF-FF, FF-solvent) increased, because of the enhanced hydrogen bond donor (HBD) ability of NH3+ compared to NH2. Meanwhile, this change in electronic interaction and hydrogen bonding was also accompanied by the enhancement of aromatic stacking between aromatic side chains, which is an important driving force for the self-assembly of FF. At 25°C, the isoelectric (pI) value of FF is approximately 5.5. In this study, the pH values of the solvents used for self-assembly were approximately 7, which is close to the pI value. The formation of FF nanowires in these solvents suggests that electrostatic and aromatic interaction may play a crucial role in the self-assembly.
Solvent parameters and the corresponding morphology of FF assemblies
Interestingly, isopropanol has a high HBA ability (0.84) capable of forming hydrogen bonds with the FF molecules. According to the mechanism previously discussed, the crystalline assemblies should form in isopropanol. However, the experimental results indicate that FF preferentially self-assembled to nanofibers and organogel during the cooling process from 90°C to 25°C. This structure remained stable and unchanged at a low temperature (-25°C) and transformed into crystalline flake or nanowire at a higher temperature (e.g., 25°C). The formation of the organogel may be attributed to both the high HBD and HBA ability of isopropanol, which allows it to form intermolecular hydrogen bonds with both amino and carbonyl groups. Hydrogen bonding between isopropanol and carbonyl groups may inhibit the formation of hydrogen bonds between the FF molecules themselves, namely, head-to-tail chains (-NH2-H…OOC-), which is a key intermolecular interaction in the formation of crystalline structures[24, 37]. Therefore, in this case, the initial FF self-assembly into an organogel was guided by other intermolecular interactions (e.g., π-π interaction). The importance of hydrogen bonds between the solvent and the carbonyl group on the supramolecular self-assembly was also demonstrated in other peptides.
Additionally, the HBA ability of isopropanol is slightly higher than the HBD ability (0.84 vs 0.76, Table 1). Similar to the solvents in group I, isopropanol should be capable of directing the self-assembly of the FF molecules to a crystalline structure with thermodynamic stability. We expected the self-assembly of the FF in isopropanol to be a kinetically controlled process. The nanofibers and organogel were formed by the fast self-assembly of the FF molecules guided by the strong π-π interaction. Furthermore, the nanofibers transformed to crystalline flakes or nanowires via the slow self-assembly as a result of the FF molecular rearrangement, which was guided by the hydrogen bonds between peptide-peptide and peptide-water molecules and the π-π interaction between aromatic side chains. The external energy, e.g., the high temperature, is used to overcome the energy barrier and speed up the ‘slow’ self-assembly process. Therefore, crystalline flake-like aggregates appeared in the organogel and uniform nanowires formed at 25°C on a glass surface. A similar kinetically controlled process was also found in the self-assembly of ferrocene-FF (Fc-FF), which initially self-assembled to metastable nanospheres and further reorganized to thermodynamically stable nanofibers with the introduction of a mechanical force. In this study, the temperature-induced structural transition from an organogel (nanofibers) to crystalline flakes and nanowires was initially found for the FF self-assembly system.
In summary, we demonstrated the temperature-triggered reversible self-assembly of diphenylalanine peptide into microtubes, nanowires, and an organogel in different solvents. In polar solvents, the dissociation equilibrium of the FF molecules was highly dependent on temperature, which likely led to the change in the electronic and aromatic interactions between the FF molecules themselves and induced the reversible self-assembly. Furthermore, we demonstrated the temperature-induced structural transition of an organogel in isopropanol to crystalline flakes and nanowires. We infer that self-assembly of the FF in isopropanol with high HBD and HBA ability, unlike in chloroform/toluene (low HBD and HBA ability), is a kinetically controlled process. The nanofibers and organogel were preferentially formed by the fast self-assembly of FF molecules guided by a strong π-π interaction and transformed to crystalline structures by introducing external energy (e.g., increased temperature) to overcome the energy barrier.
This work was supported by the Natural Science Foundation of China (Nos. 21306134, 21476165, 51173128), the 863 Program of China (Nos. 2012AA06A303, 2013AA102204), the Ministry of Science and Technology of China (No. 2012YQ090194), the Ministry of Education (No. 20130032120029), the Beiyang Young Scholar of Tianjin University (2012), and the Program of Introducing Talents of Discipline to Universities of China (No. B06006).
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