Self‐Assembly of Hexagonal Peptide Microtubes and Their Optical Waveguiding

Abstract

Hexagonal peptide microtubes are spontaneously formed upon solvent thermal annealing. They have diameters on the order of micrometers and reach several millimeters in length. The microtubes evolve hierarchically in a hexagonal packing pattern from the molecular level to the micrometer scale. Such crystalline peptide microtubes serve as an active optical waveguide, allowing for light propagation along the long axis. Self-assembly of biological building blocks including proteins and peptides is attracting increasing attention due to the versatility for bottom-up fabrication, biocompatibility and biodegradability, and a wide range of applications in biology and in nanotechnology.1 In the self-assembly process the precise control of supramolecular architectures is achieved through synergistic effects of some weak non-covalent interactions such as hydrogen bonds, electrostatic interaction, π–π stacking, hydrophobic forces, nonspecific van der Waals forces, and chiral dipole–dipole interactions.2 These weak forces, in particular hydrogen bonds and hydrophobic interactions, are ubiquitous in biological systems. As a whole they govern the molecular self-assembly into superior and ordered structures. By learning from nature, one can purposefully devise and synthesize artificial building blocks amenable to self-assembly into superstructures, which are driven by the cooperative interactions of such weak forces. With respect to other biological building blocks, peptides have been of great interest as a strategy in material fabrication owing to their structural simplicity and tunability, functional versatility, cost effectiveness, and widespread applications. As one of the simplest peptide building blocks, diphenylalanine (l-Phe-l-Phe, FF), a core recognition motif of Alzheimer's β-amyloid polypeptide,3 has been proven to be an exciting candidate towards bottom-up fabrication. FF-based building blocks are able to self-assemble into a variety of nanostructures including nanotubes, spheric vesicles, nanofibrils, nanowires, and ordered molecular chains.1 These well-defined nanostructures can be regarded as an assembly of molecules into supramolecular systems, however, organization of these into crystalline materials with spatial dimensions remains a formidable challenge. We report here that the FF building block is capable of self-assembling into robust hexagonal crystalline microtubes as a result of confined organization of nanoscale tubular structures at long range during slow crystallization or aggregation. In biological systems, the oriented organization or alignment of substructures is a common protocol to achieve functions. For instance, function of annulus fibrosus, a multilamellar fibrocartilage that forms the intervertebral (a soft tissue structure) together with the nucleus pulposus, is achieved by a high degree of structural organization over multiple length scales.4 In some sense, such hierarchical and ordered organization of substructures mimics biological systems. A distinctive tubular structure (e.g., rectangular or hexagonal structures) may provide exceptional optical, electrical, and mechanical properties that are not achievable in a circular tube.5, 6 In this work, hexagonal peptide microtubes were controllably obtained upon solvent thermal annealing. They have diameters on the order of micrometers and reach several millimeters in length. We directly observed the disassembly or debundling of peptide microtubes into nanoscale tubes by lowering the peptide concentration. This in turn demonstrates that the hexagonal hollow microtubes evolve hierarchically and consist of directionally organized nanotubes. That is, at the beginning individual dipeptide molecules self-assemble to nanotubes in a hexagonal packing pattern, which further self-organize into hexagonal microtubes through a self-similar structured growth. To the best of our knowledge, this is the first work observing hexagonal peptide microtubes that are spontaneously formed in the water phase in a hierarchical organization from the molecule level to the micrometer scale. Furthermore, such hexagonal microtubes are crystalline and can act as an active optical waveguide in conjunction with guest dyes. This finding directs a new possible way to fabricate optical materials and devices from biological or biologically derived small molecules. FF was dissolved in a small amount of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) to disrupt any aggregation that possibly occurred during peptide synthesis and purification. After removal of the HFP solvent by evaporation, the dried sample was resolubilized in ultrapure water by ultrasonic treatment for 30 min at 65 °C, and then the FF aqueous solution was annealed at room temperature. After aging for 1 day, scanning electron microscopy (SEM) was used to examine the assemblage. Surprisingly, SEM images show that the assemblages were ultralong microtubes up to several millimeters in length, with a hexagonal shape (Figure 1a and Figure S1, Supporting Information). The magnified SEM image indicates the hexagonal tubular nature and the microtube has a regular and smooth surface (Figure 1b). The SEM image of the broken end of a single microtube reveals substructures (possibly nanotubes) that are confined at long range and uniaxially oriented along the longitudinal axis (Figure 1c), reminiscent of the fiber grain in wood. a) SEM image and b) magnified SEM image of assembled microtubes. c) SEM image of the broken tube, indicating the orientationally organized substructures at long range. We found that the self-assembly of FF microtubes is concentration dependent. Disassembly of microtubes into orientationally aligned nanotubes was readily observed by using either SEM and TEM or confocal laser scanning microscopy (CLSM) when the 2 mg mL−1 microtube solution was diluted to the final FF concentration of 1 mg mL−1 with ultrapure water (Figure 2). The SEM image shows that the microtube debundles to nanotube arrays (Figure 2a), and the magnified SEM image confirms the nanotubular structures contained in the array (Figure 2b). These nanoscale tubes have sizes similar to those prepared directly at room temperature (for which a high concentration of FF/HFP solution (100 mg mL−1) was diluted to the final FF concentration of 1 mg mL−1 with water3). The TEM image reveals that the microtube splits into smaller nanotubes starting from its end (Figure 2c and Figure S2a, Supporting Information). The nanotube structure can be clearly observed in the magnified TEM image (Figure 2d). Likewise, in SEM examination, we also observed the debundling of microtubes starting from the ends (Figure S2b, Supporting Information). To directly observe the transition from microtubes to nanotube arrays in the bulk phase, FF microtubes with incorporated fluorescent dye (e.g., Nile Red (NR)) were incubated via the same strategy. It is evident that the intercalation of guest dyes into microtubes has no influence on the FF self-assembly into hexagonal shapes (Figure S3a, Supporting Information), but makes microtubes visible in a fluorescent mode under a confocal microscope (Figure S3b, Supporting Information). The dynamic transition from a single microtube into nanotube arrays was captured in the bulk phase by confocal microscopy (Figure S4, Supporting Information). It was found that debundling of the microtube started from its ends, eventually splitting into individual nanotubes (Figure 2e and Figure S4). Based on these observations, a hexagonal microtube is actually composed of a number of orientationally aligned nanotubes. This may be ascribed to the precise structural confinement achieved in the very slow crystallization process, leading to the collective organization into clusters or arrays. A verification that microtubes are composed of directionally organized nanotubes. a) SEM image and b) magnified SEM image of a debundled microtube. c) TEM image and d) magnified TEM image of a debundled microtube. e) Confocal image of a disassembled microtube starting from its end. Left: fluorescence image (original color: red); Right: bright-field image. To gain insight into the molecular interaction of microtube organization, UV-vis spectroscopy, fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy were carried out. The UV-vis spectrum of FF microtubes obtained in a reflection mode reveals a triple absorption peak centered at 257 nm, which is indicative of the aromatic residues of FF molecules (Figure 3a). No obvious shift or broadening resulted, in comparison with that of FF monomers in HFP solution, indicating that no strong π–π interactions occur between the aromatic residues.7 As shown in Figure 3b, FF microtubes have an emission peak at 285 nm with an excitation wavelength of 260 nm. Likewise, no peak shift was observed compared to free FF molecules. These results indicate that instead of π–π stacking, it is more likely that the hydrophobic collapse of FF aromatic residues becomes a driving force to direct the formation of supramolecular architectures. The appearance of hydrophobic domains in assembled microtubes was confirmed using NR as a hydrophobic probe. NR is a neutral, polarity-sensitive probe that is insoluble in water and has a maximum fluorescence emission at 650 nm in a hydrophilic environment.8 A blue-shift in NR photoluminescence (PL) (Figure 3d, 653 to 628 nm) in the presence of FF microtubes was observed. This indicates that NR molecules are incorporated into a hydrophobic domain of FF microtubes, in turn suggesting the existence of hydrophobic domains in FF microtubes.8 A slight red-shift in the fluorescence emission of FF aromatic groups (Figure 3b) in NR-incorporated microtubes is possibly ascribed to the intercalation of NR molecules into hydrophobic domains of FF microtubes, e.g., preferred to π-stacking interactions. The FTIR spectrum of microtubes (Figure S5, Supporting Information) shows the characteristic peak of the amide I band at 1607 and 1688 cm−1, corresponding to a β-sheet secondary structure.9 Intermolecular interaction for the formation of FF microtubes (FMT). a) UV-vis spectra, b) fluorescence spectra, and c) XRD patterns of FMT and FMT-NR Incorporation of NR has no effect on the packing pattern of FF molecules in the microtubes. d) Emission of NR in aqueous solution and in the FF microtube solution. X-ray diffraction (XRD) patterns of FF microtubes, including those with the incorporation of NR, are shown in Figure 3c. The diffraction pattern of microtubes is in agreement with the hexagonal structures, as determined in a study of FF single crystals10 and powder diffraction of FF nanotubes.11 Since the microtube is composed of orientationally aligned nanotubes, it is easily envisioned that they should, at molecular scale, have identical organization structures. Polarized optical microscopy (POM) images were collected to identify the optical anisotropy of FF microtubes (Figure S6, Supporting Information). Strong anisotropic PL was observed when the microtubes were placed between crossed polarizers. This suggests that hexagonal FF microtubes are birefringent, which is typically observed in crystalline materials.12 Some of the microtubes are very dark between crossed polarizers (Figure S6b). This is because their optical axis is oriented parallel to the direction of the objective polarizer, thus leading to light extinction. As the relative orientation between the sample and the crossed polarizers was rotated by 45°, the dark microtubes became bright and the originally bright ones became dark (Figure S6c). Such optical changes are consistent with uniaxially oriented alignment of FF molecules within the microtube, more likely along its long axis,11 thus leading to strong anisotropy of the refractive index.12 The formation of the hexagonal microtubes consisting of ordered nanotubes is attributed to a kinetic control of nucleation and growth during the slow crystallization.13 As reported previously, in general FF-based nanotubes prepared at room temperature by rapid dispersion of molecules from a “good” solvent into a “poor” solvent have a circular shape that is thermodynamically more stable.3, 14 The key to the formation of hexagonal hollow microtubes may be the relatively slow growth process allowing for the kinetic regulation. The slow growth leads to the confined organization of nanotubes into arrays, which undergo kinetic regulation and ultimately form hexagonal structures that are likely as a consequence of self-similar-structured growth.6, 15 As shown in Figure 4, the molecular packing occurs hexagonally. Further amplification of such packing patterns will eventually result in micrometer-sized hexagonal hollow tubes. With respect to the circular shape the hexagonal hollow structure is not the thermodynamically most stable organization, but is the kinetically most favorable form in the slow aggregation or crystallization process. We directly observed the obvious growth of microtubes in length over time, as they are discernable by optical microscopy (Figure S7, Supporting Information). Furthermore, their diameters also become larger. Apparently the microtubes do not grow by assembly of nanotubes but by growth of the assembled nanotubes. The assumption is further supported by the SEM observation of microtube growth over time (Figure S8, Supporting Information). We observed the flexible nanotube clusters as the peptide solution was annealed at room temperature for 5 min (Figure S8a). After aging for 10 min (Figure S8b), the directly organized nanotubes in a bigger tubular form were clearly observed, possibly as a growth seed for the microtube growth. With time, more organized nanotube arrays could be observed and hollow microtubes were found (Figure S8c–e). After aging for 1 day, the regular hexagonal microtubes were completely formed (Figure S8f). Schematic illustration of the formation of hexagonal peptide microtubes. FF molecules (a) first self-assemble into nanotube seeds in a hexagonal packing pattern (b–d), which can further spontaneously organize into hexagonal microtubes (e,f) by a self-similar structured growth in the process of confined crystallization. Panel (c) shows an enlarged view of the area indicated with a black rectangle in (d). These results suggest that the oriented organization of nanoscale tubes has taken place at the initial stage of hexagonal microtube formation. Then such hexagonal precursors serve as the seed for the growth of microtubes either in length or in diameter. The hexagonal microtubes are built up through a hierarchical self-assembly process. Individual dipeptide molecules initially self-assemble into nanotubes in a hexagonal packing pattern by both hydrogen bonding and hydrophobic interactions between FF aromatic residues. The confined organization and further growth of nanotubes leads to the ultimate formation of hexagonal microtubes that is kinetically favorable. Biomimetic or bioinspired structures with defined spatial dimensions are promising for the creation of photonic or electronic materials. However, few reports were found to use biological or biologically derived small molecules to serve as effective building blocks for this purpose. We found that hexagonal peptide microtubes assembled from the FF building blocks are a potential candidate for waveguide materials. In comparison to the reported organic16 or inorganic17 waveguiding materials the present dipeptide waveguide will surely extend its potential applications to biomedical fields, in which optical components are required to directly interface with living cells or systems.18 Also, provided an immune response is avoided, a peptide waveguide will naturally degrade and not leave behind any trace after it has functioned as an optical element. It is thus significantly advantageous to fabricate such functional materials through self-assembly of biologically based molecules, especially versatile self-assembling peptides. Peptide microtubes with incorporation of a guest dye (NR) were used to study optical waveguiding. PL microscopy iamges of a peptide microtube are shown in Figure 5a. The PL image indicates that the microtube has red emission as excited at 515–560 nm, resulting from the inherent NR fluorescence. Notably, there are very bright PL spots at both ends, while the tube body emits relatively weaker PL. This is a typical characteristic of an optical waveguide,16 suggesting that hexagonal peptide microtubes can act as an optical waveguiding structure. Waveguiding was further confirmed by focusing the exciting light on one end of the microtube. Clearly, the guided PL was emitted from the other end, as noted by an arrow in Figure 5a. In general the PL emission can only be observed at the local area of the excitation position. Out-coupling of light at the end is a typical waveguide behavior, which indicates that the peptide microtubes with defined spatial dimensions can serve as optical microcavities that allow for light propagation along the long tube axis. Propagation loss was studied using scanning near-field optical microscopy (SNOM) by collecting the spatially resolved PL spectra of the waveguiding emission out-coupled at the end of the microtube with incorporation of rhodamine 6G (R6G) with respect to the distance travelled (Figure 5b,c). Figure 5b shows a series of PL images of a microtube when translating the excitation spot along the microtube length from right to left. The PL intensity at the right end decreases with increasing propagation length. Figure 5c shows the dependence of the intensity of waveguiding PL out-coupled at the right end of the microtube as a function of the propagation distance. The PL intensity of the end emission decays almost exponentially with the increase in propagation length, indicating a typical active waveguide. The waveguide losses in the peptide microtube can be attributed to the effect of the substrate directly in contact with a facet of the microtube and to Rayleigh scattering caused by possible compositional inhomogeneities and structural defects.16 Optical waveguiding of hexagonal peptide microtubes with incorporation of guest dyes (NR or R6G). a) PL microscopy images of a single microtube excited at 515–560 nm. Left: bright-field image; Middle: PL image; Right: direct observation of waveguiding with local excitation at one end. The light blue circle marks the excitation area and the green arrow denotes the out-coupling of PL emission at the other end. b) Bright-field image and PL images of a single microtube in a SNOM measurement. PL images showing optical waveguiding upon excitation of the identical microtube at different positions. c) Spatially resolved PL spectra of waveguided emission at the end of a microtube. The end intensities were normalized to those measured at the excitation position in the body of the microtube. Inset shows Iout/Iin vs excitation position for the PL spectra and exponential fit (red line). Iout is the out-coupled PL intensity at the end and Iin excitation PL intensity in the body. In summary, we have reported the spontaneous formation of hexagonal peptide microtubes in the bulk phase using a solvent thermal annealing process. It has been demonstrated that the microtubes evolve hierarchically in a hexagonal packing form from the molecular level to the micrometer scale. Kinetic control of nucleation and growth is suggested to be necessary for the ordered organization of substructures in the microtubes. Such crystalline peptide microtubes with hexagonal dimensions exhibit active optical waveguiding, a physical property newly emerging in bioinspired small molecules. This finding may direct photonic and electronic devices towards peptide-based biomolecular materials. Preparation of Hexagonal Peptide Microtubes: The lyophilized diphenylalanine (H-Phe-Phe-COOH, FF) (Bachem, Bubendorf, Switzerland) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP, Sigma-Aldrich) to disrupt any possible aggregation that occurred in the peptide sample. The FF/HFP solution was allowed to dry by evaporation under ambient conditions. By addition of an aliquot of ultrapure water and subsequent ultrasonic treatment at 65 °C for 30 min, FF was resolubilized in water. The FF aqueous solution was then immediately annealed at room temperature and allowed to age for one day. The obtained assemblage was used for further characterization. Microscopy: SEM measurements were performed using a Philips XL30 electron microscope at an accelerating voltage of 3 kV. TEM images of crystalline platelets were recorded using a Zeiss EM 912 Omega transmission electron microscope operated at 120 kV, with samples carefully placed onto the carbon-coated copper grids. Confocal laser scanning microscopy (CLSM, Leica TCS SP) was used to monitor the dynamic disassembly of NR incorporated microtubes. Excitation source was a 543 nm HeNe The growth of a microtube along the long axis was directly observed by an 2 optical microscope with a of FF aqueous solution was onto a and allowed to to room temperature. were taken as a function of time when tubes were PL and optical waveguide were obtained using a Leica microscope with a Propagation loss of the peptide microtube waveguide was carried using a scanning near-field optical with a nm laser was as the excitation UV-vis spectra were recorded on a with an for the of samples in reflection A was used to the PL of peptide microtubes in water in a XRD patterns were collected at room temperature using a X-ray with The FTIR spectrum was recorded on a by of Supporting is from the or from the from a of the The the of this by the of and the of as as the and for SEM and TEM and are for their with SNOM of to are as Such are but not or They are as by the The is not for the or of any by the be to the corresponding for the