Article pubs.acs.org/Langmuir
The Rheological and Structural Properties of Fmoc-Peptide-Based Hydrogels: The Effect of Aromatic Molecular Architecture on SelfAssembly and Physical Characteristics Ron Orbach,† Iris Mironi-Harpaz,‡ Lihi Adler-Abramovich,† Estelle Mossou,§,∥ Edward P. Mitchell,∥,⊥ V. Trevor Forsyth,§,∥ Ehud Gazit,† and Dror Seliktar*,‡ †
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Science, Tel-Aviv University, Tel-Aviv 69978, Israel ‡ Faculty of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel § Partnership for Structural Biology, Institut Laue Langevin, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9, France ∥ EPSAM/ISTM, Keele University, Staffordshire ST5 5BG, U.K. ⊥ ESRF Experiments Division, 6 rue Jules Horowitz, 38043 Grenoble Cedex 9, France ABSTRACT: Biocompatible hydrogels are of high interest as a class of biomaterials for tissue engineering, regenerative medicine, and controlled drug delivery. These materials offer three-dimensional scaffolds to support the growth of cells and development of hierarchical tissue structures. Fmoc-peptides were previously demonstrated as attractive building blocks for biocompatible hydrogels. Here, we further investigate the biophysical properties of Fmoc-peptide-based hydrogels for medical applications. We describe the structural and thermal properties of these Fmoc-peptides, as well as their self-assembly process. Additionally, we study the role of interactions between aromatic moieties in the self-assembly process and on the physical and structural properties of the hydrogels.
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INTRODUCTION The fabrication of molecular biomaterials can be achieved by two complementary strategies.1 In the “top-down” approach biomaterials are generated by “sculpturing” a complex entity into the desirable component parts. By contrast, the “bottomup” approach is based on molecular self-assembly, where biomaterials are assembled molecule by molecule, through noncovalent interactions, to produce well-ordered ultrastructures. In the latter process, the structure of the molecular building blocks determines the architecture of the entire assembly.2 The self-assembly process is usually mediated through weak intermolecular interactions, such as van der Waals interactions, hydrogen bonds, aromatic interactions, and electrostatic interactions. Short peptides, as well as proteins and large polypeptides, can self-assemble into various nanostructures such as spheres, tubes, and tapes.3−11 Diverse chemical and structural species integrated into these short peptides may confer upon them some advantages over other building blocks in the construction of complex architectures.3,12−15 These nanostructures can form unique materials at macroscopic as well as nanoscopic scales, including nanoscale ordered hydrogels.16−20 Hydrogels are frequently used as three-dimensional (3D) scaffolds to support the growth of cultured cells for tissue engineering and regeneration.21−23 They are appealing for © 2012 American Chemical Society
various medical uses owing to their similarity to the natural extracellular matrix (ECM), which allows cell adhesion via specific peptides, such as Arg-Gly-Asp (RGD), while having very good biocompatibility.24 Peptide-based hydrogels exhibit the advantages of both synthetic and naturally derived hydrogel-forming materials. The peptides are easy to manufacture in large quantities and can also be decorated both chemically and biologically with ease. Such decoration enables the design of ultrastructures with ligand moieties, as well as other biologically functional groups, that may allow cell adhesion and growth.25,26 Furthermore, the self-assembly of short peptides with an aromatic nature is of central biological importance because nuclear pore complexes are often endowed with a permeability barrier that contains short clusters, which contains aromatic amino acids, such as Phe-Ser-Phe-Gly (FSFG). It has been suggested that these clusters form 3D interwoven networks having hydrogel-like properties, which are implicated in their selectivity.27 Thus, in order to gain insight into both natural and synthetic peptide-based hydrogel structures, an understanding of the self-assembly process of short aromatic peptides is sought. Special Issue: Bioinspired Assemblies and Interfaces Received: November 10, 2011 Published: January 5, 2012 2015
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Figure 1. Molecular structure of the Fmoc-peptides.
aromatic moieties to the length of Fmoc-peptide backbone, and in the position of the additional aromatic moiety.
In the past few years there has been a keen interest in Fmocpeptides and their various applications.28−37 In 1995 Vegners et al. reported for the first time the formation of fibrous networks with fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and dipeptides.38 Burch et al. reported that a number of Fmoc amino acids also exhibit anti-inflammatory properties.39 We previously reported on the efficient self-assembly, under mild conditions, of the Fmoc-Phe-Phe (Fmoc-FF) peptide into a rigid hydrogel with remarkable physical properties.40 In this work, we speculated that aromatic interactions may play a key role in the formation of tubular structures, as they contribute to the free energy of formation, and impart order and directionality to the self-assembly process. Parallel independent work by Ulijn et al. demonstrated the spontaneous assembly of additional Fmoc-peptides into fibrous hydrogels, and the effect of pH on the self-assembly process was investigated.28,33,41 Our hypothesis was also supported by their work, which employed spectroscopic techniques to demonstrate that Fmoc-FF forms fibrous antiparallel β-sheet structures.42 Recently, we expanded our findings to include additional members of the aromatic Fmoc-dipeptide family, and demonstrated molecular selfassembly with a new set of Fmoc-peptides, which included both natural and non-natural aromatic amino acids. We examined self-assembly into various nanoscale structures and characterized the distinctive molecular and physical properties of these assemblies. We identified a subset of these Fmocpeptides that formed 3D interwoven fibrillar networks through branching and flexible primary structures with a diameter ranging from 10 to 30 nm that absorb and retain water under physiological conditions.43 In the present study, we expand upon our previous work by investigating the molecular self-assembly process during gelation of five Fmoc-peptides: Fmoc-FF, Fmoc-Phe-Gly (Fmoc-FG), Fmoc-β-(2-naphthyl)-L-alanine (Fmoc-2-Nal), Fmoc-Phe-Arg-Gly-Asp (Fmoc-FRGD) and Fmoc-Arg-GlyAsp-Phe (Fmoc-RGDF) (Figure 1). These specific peptides were chosen in order to investigate the role of the aromatic composition in the self-assembly process. To this end, all the peptides contained a Fmoc group modification at their Nterminus. All peptides also contained at least one additional aromatic moiety, in addition to the Fmoc group, based on the rationale that aromatic interactions are essential to the selfassembly process.44−51 The peptides varied both in the ratio of
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MATERIALS AND METHODS
Peptides. Lyophilized peptides Fmoc-FF, Fmoc-2-Nal, and FmocFG were purchased from Bachem (Bubendorf, Switzerland) and used without further purification. Fmoc-FRGD and Fmoc-RGDF were HPLC-purified (≥ 95%) and synthesized by Peptron (Daejeon, South Korea). Preparation of Hydrogels. Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) (Merck) for peptide solutions at concentrations of 100 mg/mL, and the hydrogels were prepared by diluting the stock solution in ultra pure water (Biological Industries, Beit Haemeck, Israel). Various hydrogel concentrations were prepared by using different peptide-solution-to-water ratios. To avoid any preaggregation and assembly, fresh stock solutions were prepared for each experiment. Rheology. The in situ hydrogel formation, mechanical properties, and cross-linking kinetics were characterized by an AR-G2 rheometer (TA Instruments). Time-sweep oscillatory tests in 20 mm parallelplate geometry were performed on 210 μL of fresh solution (resulting in a gap size of 0.6 mm), 1 min after its preparation, at room temperature. Each Fmoc-peptide was tested three times, and their average is shown. Oscillatory strain (0.01−100%) and frequency sweeps (0.01−100 Hz) were conducted 10 min after diluting the stock solution in water in order to find the linear viscoelastic region, at which the time sweep oscillatory tests were performed. The linear viscoelastic region was found to differ between hydrogels. Thus, for the different peptides, the rheological tests were performed at different conditions (i.e., Fmoc-FF: 0.6% strain, 10 Hz frequency; Fmoc-RGDF: 1% strain, 4 Hz frequency; Fmoc-FRGD: 0.2% strain, 0.5 Hz frequency; Fmoc-2Nal: 0.6% strain 1 Hz frequency; Fmoc-FG: 0.7%strain, 1 Hz frequency). Turbidity Analysis. Hydrogel peptide solutions were prepared in test tubes at concentrations of 1, 2.5, and 5 mg/mL. A 50 μL aliquot was pipetted into a 96-well plate (nunc) and absorption kinetic was measured at 25 and 37 °C at 405 nm, using Varioskan (Thermo Electron Corporation), starting 40 s after the preparation of the peptide solution. The results of each experiment and their comparison were normalized separately. Fiber Diffraction. Experiments were performed at the ID14-2 beamline of the European Synchrotron Radiation Facility (ESRF). The wavelength was 0.933 Å, and the beam was around 90 μm with a square cross-section. The experiments were performed using the ID142 microgoniometer at room temperature. Data were recorded using an ADSC Q4 CCD-based detector with 145 mm converter screen (2048 × 2048 pixels, 16 bit readout). The sample-to-detector distance was
2016
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calibrated by an Ag-behenate standard. Diffraction data sets were processed and measured using CCP13 software and the FIT2D package. Thermogravimetric Analysis (TGA). Thermogravimetric tests were carried out with a TA Instruments (USA) module SDT 2950. Analysis was conducted isothermally for 30 min at room temperature and afterward in a scan rate of 10 °C/min up to 250 °C, under dried, ultrahigh-purity argon atmosphere. The measurements started 70 s after the solution formation.
that the elastic response component (G′, storage modulus), which was initially lower than the viscous response component (G″, loss modulus, not shown) exceeded it with time, by at least 1 order of magnitude, indicating a phase transition and the formation of a hydrogel polymer network. Generally, similar hydrogel formation kinetic patterns showing two sequential shoulders were observed for the different concentrations of Fmoc-2-Nal (at a constant temperature of 25 °C). The plateau G′ values increased, and the time required to reach the plateau G′ values, as well as the lag time between the two consecutive shoulders, decreased with increasing concentrations (Figure 2a). However, when the elastic properties of the hydrogels were investigated at different temperatures and at constant peptide concentrations (e.g., 5 mg/mL), they exhibited various profiles of hydrogel formation kinetics (Figure 2b). Spectroscopic Characteristics. Optical measurements of the self-assembly kinetics serve as a useful tool that strongly complements rheological kinetic analysis. It enables one to follow the self-assembly process and the formation of structures with dimensions at the range of the visible wavelength, along with the comparison of the change in rheological properties. The dilution of the peptide−DMSO solutions with water leads to a unique phenomenon, as it forms a turbid viscous solution, which subsequently alters the transparency of the developing hydrogel. Spectroscopic techniques were therefore used to study the influence of the peptide concentration and temperature on the hydrogel formation kinetics. All of the peptides except Fmoc-FRGD exhibited a similar kinetic pattern of hydrogel formation at the tested concentrations and temperatures (Figure 3). The peptide solutions displayed higher initial turbidity values and a faster decrease in turbidity with increasing peptide concentrations at both 25 and 37 °C. Additionally, the turbidity decreased as a function of the solution temperature. The formation of the Fmoc-FRGD peptide into a hydrogel resulted in a different kinetic pattern altogether, first exhibiting an increase in turbidity followed by a significant decrease in turbidity (Figure 3d). The other peptide solutions exhibited only a decrease of the turbidity over time. Interestingly, the turbidity of the Fmoc-FRGD remained constant at concentrations of 1 and 2.5 mg/mL at 25 °C; a comparison with rheological data revealed that at these conditions the Fmoc-FRGD solution did not form a hydrogel. Figure 4 shows a comparison of the appearance of different peptide solutions (5 mg/mL) at two different temperatures. It is worth noting that due to the rapid formation of Fmoc-FG (30 s), its optical transition kinetics were not documented and unlike the other tested materials, this material was unstable and eventually precipitated out of solution.41 Hybrid Hydrogels. Hybrid hydrogels were formed by mixing two different peptides at a 1:1 concentration ratio. Hybrids were made with Fmoc-FF, Fmoc-2-Nal, and Fmoc-FG peptides due to their rapid gelation. Although Fmoc-FG forms an unstable hydrogel by itself, the mixture of Fmoc-FG with Fmoc-FF formed a stable hybrid hydrogel, which required prolonged periods of time for the optical transition to culminate (approximately 60 min). By contrast, the optical transitions for the individual peptides took place after 3.5 min and 30 s for Fmoc-FF and Fmoc-FG, respectively. This interesting phenomenon was also observed with the Fmoc-FF and Fmoc-2-Nal mixture. These observations were further supported by the spectroscopic analysis of the Fmoc-FF with Fmoc-FG mixture, tested at different concentrations and temperatures (summarized in Figure 5). The spectroscopic
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RESULTS Rheological Properties. One of the best methods to determine the rate of the self-assembly process in hydrogel formation is time-dependent rheology. Analysis of the changes in the material’s viscoelastic properties over time, as well as the material’s final complex shear modulus, is often used to understand phase transitions associated with molecular rearrangement in aqueous environments. Here, we used timedependent rheological analysis to study the role of aromatic moieties in the self-assembly process of the Fmoc-peptides. Accordingly, we studied the influence of peptide concentration and temperature on the viscoelastic properties of the hydrogels being assembled, and on the kinetics of the hydrogel formation. The Fmoc-peptides were tested for their rheological properties, and Fmoc-2-Nal was chosen as a representative model (Figure 2). The complex shear modulus (G*) of the hydrogels showed
Figure 2. Temperature and concentration effect on Fmoc-2-Nal hydrogel formation kinetics. (a) Rheological analysis of the hydrogels at various concentrations at 25 °C exhibits a similar kinetic pattern for each of the hydrogels, excluding time shift and G′ plateau. (b) Rheological analysis of the hydrogels at a concentration of 5 mg/mL exhibits different kinetic patterns, due to the faster self-assembly as the temperature increases. 2017
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Figure 3. Turbidity changes in the hydrogel formation. (a) Fmoc-FF, (b) Fmoc-2-Nal, and (c) Fmoc-RGDF show the same absorption change pattern, as it starts with high and steady absorption (opaque) which changes into exponential decrease and ends with low and steady absorption (transparence). (d) Fmoc-FRGD exhibits a different pattern, as it starts with absorption elevation. All the peptide solutions were examined at different concentrations and at 37 °C (solid curves) and 25 °C (dashed curves).
Figure 4. Spectroscopic analysis comparison. A comparison of optical change of the hydrogels Fmoc-2-Nal, Fmoc-FF, Fmoc-FRGD, and Fmoc-RGDF at a concentration of 5 mg/mL at 25 °C or at 37 °C.
Figure 5. The turbidity changes in the hybrid hydrogel. The absorption changes of the Fmoc-FF and Fmoc-FG mixture show longer self-assembly time than any other single peptide. However, it behaves in the same manner as the other single peptides regarding temperature and concentration influence.
analysis showed that the mixture had a similar behavior to that of the individual peptides, i.e., the initial turbidity was higher, and the optical transition was faster with increasing concentration and temperature. However, the kinetic pattern of the hydrogel formation process was different from those of the individual peptides, showing maxima in absorption as the hydrogel is formed. This phenomenon was also studied at different concentration ratios of peptides in order to understand whether this affected hydrogel formation kinetics. The slowest optical transition was observed at a 1:2 ratio of FmocFG:Fmoc-FF. As the ratio of one of the peptides increased, so did the transition time (Figure 6). The rheological data recorded from the Fmoc-FF mixed with Fmoc-FG (at a 1:1 ratio) also showed that the hydrogel selfassembly kinetics and viscoelastic properties were different for
the mixed solutions, in comparison with the individual Fmocpeptides (Figure 7). The results show that the initial G′ value for the mixture was much lower than that of the individual peptides. Moreover, the increase in G′ continued even after 45 min, while the individual peptides already showed a plateau at the same time point. Hydrogel Stability. There is sufficient experimental data to support the theory that the aromatic composition of the peptides influences the self-assembly process as well as the bulk properties of the hydrogels. In fact, a comprehensive study that compared thermophilic proteins to their mesophilic homo2018
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peptide was used as a control that does not contain any aromatic moieties and which is unable to form ordered
Figure 6. Effect of concentration ratios on the turbidity of an Fmoc-FF and Fmoc-FG mixture. Different concentration ratios showed that the slowest optical transition was observed at a 1:2 ratio of FmocFG:Fmoc-FF.
Figure 8. TGA. (a) The weight loss of the different hydrogels measured as a function of the temperature elevation. (b) Derivative weight shows the stability of the hydrogels. (c) The weight of FmocFRGD and AA at two different concentrations measured at increasing temperature.
nanoscopic structures that allow the incorporation of water molecules. The weight derivative of the TGA curves demonstrated that the weight loss of Fmoc-FRGD and Fmoc-RGDF began at ∼50 °C. Although both of these hydrogels exhibited weight loss at lower temperatures than AA, the weight loss was moderate and reached a peak at ∼120 °C, while AA weight loss reached a peak at ∼100 °C (Figure 8b). Accordingly, the total weight loss of the AA solution was at ∼140 °C, while all the hydrogels exhibited a total weight loss at ∼160 °C (Figure 8a). The Fmoc-FF and Fmoc-2-Nal peptides displayed higher thermal stability, compared to Fmoc-FRGD and Fmoc-RGDF, with minor weight loss up to ∼110 °C (Figure 8a). The effect of the peptide concentration on the
Figure 7. Rheology tests of hybrid hydrogels. Rheology tests of the peptides and the mixtures in the ratio of 1:1 show a significantly different kinetic pattern of the mixture from both peptides.
logues demonstrated the importance of aromatic clusters in the context of increased thermal stability.52 We have previously demonstrated that the aromatic moieties on the Fmoc-peptides have a profound influence on the ultrastructure of the resulting hydrogel network.43 TGA was therefore applied to monitor the weight loss with increasing temperature of the various hydrogels. The weight loss due to water evaporation enabled us to determine the thermal stability of the Fmoc-peptide hydrogel structures (Figure 8). The H-Ala-Ala-OH (AA) 2019
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Figure 9. X-ray fiber diffraction. Diffraction patterns were recorded for (a) Fmoc-FF, (b) Fmoc-2-Nal, (c) Fmoc-FRGD, and (d) Fmoc-RGDF. These patterns show the diffraction features associated with the cross- β structure.
diffraction pattern recorded from Fmoc-2-Nal showed a rather more crystalline pattern (Figure 9b) for which the meridional reflection is observed at 5 Å. While a 5 Å feature of this type is unusually high for cross-β fiber diffraction patterns, such values have been observed previously.54,55 The X-ray diffraction experiments, in combination with previous FT-IR, electron microscopy, and Congo Red staining results, show that these molecular structures adopt a cross-β structure of the type that is commonly observed in amyloids.43 Indeed, these observations are collectively taken as a diagnostic of a cross-β structure in amyloid work. The hydrogel structures are therefore assumed to consist of β-strands running perpendicular to the fiber axis, forming stacked hydrogen-bonded sheets with a sheet spacing that varies somewhat between the peptides but is typically ∼10 Å. All of the information available so far suggests that water is very tightly bound in these structures, and it is therefore assumed that much of it is highly structured. The results from this work are therefore of further interest in the context of hydration in amyloid-type systems.
hydrogel thermal stability was also examined. The AA peptide that was used as a control and Fmoc-FRGD showed better thermal stability at high concentrations, exhibiting a slower weight loss. However, the TGA curves showed minimal weight loss for Fmoc-FRGD up to a temperature of ∼85 °C, while AA peptide’s weight loss began around the same temperature as it did at a lower concentration (Figure 8c). The TGA results indicated that Fmoc-FF and Fmoc-2-Nal have significantly higher thermal stability than Fmoc-FRGD and Fmoc-RGDF peptides (Figure 8). Examination of their molecular structure shows that Fmoc-FF and Fmoc-2-Nal both comprise a 1:1 aromatic moieties ratio, while Fmoc-FRGD and Fmoc-RGDF both comprise a 2:5 aromatic moieties ratio. These results imply that higher aromatic moieties ratio on the peptide backbone may provide higher thermal stability.52 Secondary Structure. Fiber diffraction studies were carried out in order to probe certain specific aspects of the structure and regularity of these peptide filaments. The diffraction studies were carried out using shear-aligned samples in which a high degree of the filamentous material is rendered parallel to the axis of a macroscopic sample. This type of study provides direct information on molecular structure. The diffraction patterns recorded from the Fmoc-FF, Fmoc-2-Nal, Fmoc-FRGD, and Fmoc-RGDF fibers are shown in Figure 9. Each of these diffraction patterns show the characteristic features of a cross-β structure, with a sharp well-defined meridional reflection corresponding to a periodicity of 4.7 Å. This feature is normally taken to arise from the axial separation of hydrogenbonded β-strands making the β-sheet. Furthermore, the patterns also demonstrated clear, although more diffuse, periodicities of approximately 10 Å in the equatorial direction − believed to arise from the stacking of the β-sheets in the structure. This periodicity is more variable, and is likely to depend on the side-chain content of the peptide.53 The
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DISCUSSION The self-assembly of macromolecules can be generally characterized by an optical transformation from a transparent solution to a turbid solution, or hydrogel, in the range of 313− 500 nm. This transition is characterized by a sigmoid curve, which contains lag, growth, and plateau phases. It was suggested that the nucleation process occurs during the lag phase, which is rapidly extended during the growth phase.56 In the current work, we observed the opposite phenomenon of an optical transition as the formation of the hydrogel is accompanied by a transition from a turbid solution to a transparent network. This optical change was monitored by spectroscopic analysis, demonstrating similar kinetic patterns for Fmoc-FF, Fmoc-2-Nal, and Fmoc-RGDF peptides. All three 2020
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hypothesis is supported by spectroscopic analysis. Thus, a turbidity comparison between the Fmoc-peptides showed that a longer amino acid sequence and the presence of less hydrophobic moieties, such as Fmoc-FRGD and FmocRGDF, exhibited lower turbidity compared to shorter and more hydrophobic Fmoc-peptides, such as Fmoc-FF and Fmoc-2-Nal. Additionally, the spectroscopic analysis of FmocFRGD at a concentration of 1 mg/mL at both 25 and 37 °C and at a concentration of 2.5 mg/mL at 25 °C demonstrated no optical changes during the self-assembly process (Figure 3d). When rheological testing was performed to verify self-assembly, we discovered that the peptides did not form a hydrogel under these conditions (data not shown). When exploring the behavior of a mixture of two different peptides, a slower process was exhibited by the mixture in comparison to the individual peptides. This was confirmed by both spectroscopy and rheological analyses of measuring self-assembly kinetics. The results are consistent with the rationale that the intricacies involved in forming more complex ultrastructures made from the two types of peptides significantly decelerate the process of self-assembly. Alternatively, the diffusional effects involved in the coformation of two distinct populations of structures may also slow down the self-assembly process. Irrespective of the exact kinetics of self-assembly, a comparison of the rheological results and the spectroscopic analysis (Figures 2 and 3) evidently points to the fact that the optical transition of the various hydrogels occurs during the exponential phase of their elasticity transition. Thus, the fibril formation phase of the selfassembly process may be associated with the largest and most significant increase in the observed material’s elastic properties (G′).
peptides initially formed turbid solutions that turned into transparent hydrogels after the self-assembly process culminated (Figure 3a−c). However, Fmoc-FRGD peptide (Figure 3d) and the mixture of Fmoc-FF with Fmoc-FG (Figure 5) exhibited different patterns, as the turbidity initially increased and only then decreased to a transparent hydrogel network. We speculate that the increase in turbidity occurs in all the peptide solutions, but due to relatively fast kinetics it cannot be monitored spectroscopically using our experimental setup. Additionally, comparison of the gelation process at 25 °C suggested that increasing the aromatic ratio on the peptide backbone (Fmoc-2-Nal > Fmoc-FRGD) results in a faster rate of the self-assembly process, probably due to the elevation in hydrophobicity of the peptide molecules (Figure 4). Interestingly, the spectroscopic analysis at 37 °C revealed that the peptide self-assembly formation rate was altered for all the concentrations studied (Fmoc-2-Nal > Fmoc-RGDF > FmocFF > Fmoc-FRGD), a result that was further supported by rheological testing (data not shown). Therefore, it appears that other parameters may also significantly affect the self-assembly process. We hypothesized that the duration of the optical transition in the Fmoc-peptide and therefore the self-assembly process corresponded to the time required for the molecules in their initial organization to undergo a physical restructuring. This restructuring from many irregular aggregates, having dimensions in the range of the visible wavelength, into highly ordered structures causes the optical characteristics of the solution to change, and thus can be used to measure the kinetics of the self-assembly process. Presumably, the final diameter of the self-assembled structure is much smaller than the visible wavelength (10−30 nm), thereby forming a hydrogel with transparent optical properties (Figure 10).
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CONCLUSIONS Interactions between aromatic moieties have a significant contribution on the self-assembly process of a class of extremely short peptides that form stable hydrogels of remarkable physical properties. The aromatic structural elements contribute free energy of formation as well as order and directionality to the self-assembly process. We demonstrated the significance of aromatic residues in the self-assembly process of Fmoc-peptides by showing that similar peptides having different configurations of aromatic residues exhibited significantly different hydrogel formation kinetics. The results have provided new insights into the pivotal role that the aromatic features of each peptide have on the self-assembly process itself (i.e., the ratio and location of the aromatic moieties on the peptide backbone). By comparing a variety of Fmoc-peptides, we revealed the critical role of the aromatic groups in the regulation of the self-assembly process and consequently their influence on the structural and physical properties of the formed hydrogels. We have previously shown, using electron microscopy, the influence of the number of aromatic moieties and their position on the peptide backbone on the morphology of the ultrastructures. It also appeared that a higher aromatic ratio caused higher yield of structures. The TGA demonstrated the aromatic contribution to the thermal stability of the formed hydrogels. The aromatic influence on the self-assembly kinetics was shown by the rheological and spectroscopic analyses, i.e., higher aromatic ratio resulted in faster self-assembly kinetics. Finally, it was demonstrated that as the number of aromatic groups on the peptide backbone increased, the elasticity of the hydrogel is increased correspondingly.
Figure 10. Turbidity−time curve illustrating lag phase, during which irregular aggregates form, accompanied by small turbidity increase, and growth phase, during which fibers form and rapidly grow into crystalline fibers. The plateau is characteristic of termination of fibril growth.
The Fmoc-peptides comprise a high number of aromatic groups exhibiting a hydrophobic nature, which decelerate their solvation in water. Therefore, the peptides were first dissolved in a hydrophobic solvent forming a solution of monomers. The transition from the solvent hydrophobic environment into the water hydrophilic environment results in monomer insolubility, which is expressed by high turbidity of the solution. This 2021
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
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ACKNOWLEDGMENTS E.G. and V.T.F. thank the European Commission framework program for financial support (BeNatural/NMP4-CT-2006033256). We thank Dr. Diana Golodnitsky for TGA experiments and all the members of the laboratories for helpful discussion.
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