Regular Article
Control of peptide hydrogel formation and stability via heating treatment

https://doi.org/10.1016/j.jcis.2020.09.032Get rights and content

Highlights

  • Stable peptide hydrogels can be assembled via thermal treatment under kinetic control.

  • Thermo-induced chiral inversion was observed during the sol-gel transition.

  • Repeated heating and quenching cycles can improve the stability of the hydrogel.

Abstract

Heating treatment is widely used in the preparation of metallic materials with controlled phase behavior and mechanical properties. However, for the soft materials assembled by short peptides, especially simple dipeptides, the detailed influences of heating treatment on the structures and functions of the materials remain largely unexplored. Here we showed that by thermal annealing or quenching of aromatic peptide solutions under kinetic control, we are able to control the self-assembly of peptide into materials with distinct phase behavior and macroscopic properties. The thermal annealing of the heated peptide solutions will lead to the formation of large nanobelts or bundles in solution, and no gels will be formed. However, by quenching the heated peptide solution, a self-supporting hydrogel will be formed quickly. Structure analysis revealed that the peptides preferred to self-assembled into much thinner and flexible nanohelices during quenching treatment. Moreover, the stability of the gels further increased with the repeated heating and quenching cycling of the peptide solutions. The results demonstrated that the heat treatment can be used to control the structure and function of self-assembled materials in a way similar to that of the conventional metallic or alloy materials.

Graphical abstract

The Fc-FF hydrogels were fabricated by adjusting the heating treatment methods, and the multiple heating and quenching cycles were contributed to improving the stability of hydrogels.

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Introduction

Self-assembly of biologically relevant molecules such as peptides is of great significance for many fields, including biomedicine [1], [2], catalysis [3], biosensors [4], and chiral optics [5] due to their biocompatibility and structural diversity. Peptides are small biomolecules composed of a series of amino acids, which can self-assemble to form biological materials, such as gels and liquid crystals [6], [7], [8]. Moreover, peptides can self-assemble into diverse nanostructures such as nanofibers [9], nanotubes [10], nanoarrays [11], etc. Currently, many studies have discussed the self-assembly pathway of peptides [6], [12], [13], [14]. On one hand, the thermodynamic driving forces responsible for peptide self-assembly are non-covalent interactions, such as π-π stacking, hydrogen bonding, hydrophobic, and electrostatic interactions [15], [16], [17], which is the main driving force of the self-assembly process. On the other hand, the kinetic parameters including time and temperatures also have great influences on the self-assembly of the peptides to form different nanostructures [12], [18], [19], [20], which was demonstrated in several studies that the structure of the assemblies could be transformed under kinetics control [21], [22]. For example, Stupp group [23] explored the assembly pathway of peptide amphiphile (PAs) by changing solvent-composition. The results demonstrated that supramolecular morphologies of PAs could be strongly affected by different kinetic factors like hysteresis or the influence of solvent composition.

Short aromatic peptide derivatives have been proven to be good hydrogelators [24], [25]. The diphenylalanine (NH2-l-Phe-l-Phe-COOH, FF) is one of the smallest peptide building blocks, which is extracted from the Alzheimer’s β-amyloid polypeptide [26]. The phenomenon of the structural transition of FF-based peptide from organogel to crystals by changing various kinetic parameters have been observed in several systems, including temperature, solvent, and sonication [27], [28], [29]. Adams et al. [20] have shown a simple heat/cool cycle could significantly affect the properties of a solution of a low-molecular-weight gelator (2NapFF). In our previous work [30], we reported the structural transition (from metastable nanospheres to well-defined nanofibers) of the ferrocene-modified derivative of FF (Fc–FF) by introducing a mechanical force under kinetic control, and the self-assembly of the Fc–FF could be reversibly controlled by the redox center of the ferrocene group. However, the influence mechanism of heating treatment on soft materials assembled by short peptides remains largely unexplored.

In the metallurgy or materials engineering, the heating treatment process is widely used to change the mechanical properties, such as hardness and strength of materials [31], [32], [33], [34]. The thermal quenching and annealing are common heating treatment methods. Specifically, the quenching can enhance the strength and hardness of the materials by rapidly cooling. On the other hand, the annealing is to change the microstructure of the materials to increase the flexibility, ductility, and reduce the hardness. It is worth noting that there have been a few reports on the annealing treatment of peptide-based systems and other low molecular weight gelling systems [13], [35], [36], [37], [38]. For example, Stupp [13] studied the effects of dilution and annealing on the amphipathic structure of peptide amphiphiles and chromophore amphiphile. Adams et al. [35] reported that annealing multicomponent gels could be used to prepare materials with tunable mechanical properties. The annealing affected both the absolute stiffness and also the breakage strain. Here, we explored the chiral self-assembly of short aromatic peptides in isopropanol/water mixture solutions, and plotted a phase diagram for the self-assembly of Fc-FF as a function of water content and temperature, which illustrated three distinct self-assembling regimes. Interestingly, the heating treatment led to the formation of Fc-FF hydrogels by adjusting the cooling rate. The circular dichroism (CD) spectroscopy and scanning electron microscopy (SEM) have been used to characterize the hydrogels structures. Moreover, it was demonstrated that multiple heating and quenching cycles could improve the stability of the Fc-FF hydrogel. The results indicated that the self-assembling kinetics of Fc-FF have a significant influence on controlling the formation of stable hydrogels. Finally, the versatility of the heating treatment method with improving the mechanical properties of hydrogels was demonstrated during the self-assembly of a N-(9-fluorenylmethoxycarbonyl)-protected phenylalanine-phenylalanine-aspartic tripeptide (Fmoc-FFD).

Section snippets

Chemicals and materials

Fc-FF was synthesized according to our previous work [30]. NH2-l-Phe-l-Phe-COOH (FF) (98% in purity) peptides were purchased from Bachem (Bubendorf, Switzerland). Ferrocene carboxylic acid was purchased from J&K Scientific Ltd. (Beijing, China) (98% in purity). O-(Benzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium tetrafluoroborate (TBTU) was purchased from Alfa Aesar (Shanghai, China) (98% in purity). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was purchased from Aladdin Reagent Corporation

Phase diagram for the gelation of Fc-FF in water/isopropanol mixture solvent

To probe the gelation behavior of Fc-FF peptide, the lyophilized Fc-FF power was dissolved in isopropanol/water mixture solutions, then diluted in pH 5.9 phosphate buffer (100 mM) to give a final concentration of 2 mg mL−1 and incubated for 1 h at 20 °C. Scanning electron microscopy (SEM) images revealed that the morphology of the Fc-FF peptide self-assembled into nanospheres when the water content (C = volwater/volisopropanol+water) was 10% (Fig. 1a). When the C was increased to 70%, the

Conclusion

Compared with the reported methods for controlling the formation of the hydrogels [24], [25], [56], [57], this work has demonstrated that short aromatic peptides could self-assembled into the stable hydrogels via a simple heating treatment. Firstly, the repeated heating and quenching cycling of the peptide solutions was the simple and efficient method to obtain stable hydrogel, which was attributed to the process of kinetic control, whereas the thermal annealing of the heated peptide solutions

CRediT authorship contribution statement

Qing Li: Methodology, Investigation, Writing - original draft. Gong Zhang: Methodology, Investigation. Yifei Wu: Methodology. Yuefei Wang: Conceptualization, Supervision, Investigation, Writing - review & editing. Yaoyu Liang: Methodology. Xin Yang: Methodology. Wei Qi: Supervision, Funding acquisition. Rongxin Su: Visualization, Investigation. Zhimin He: Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 51773149, 21621004, 22078239), the Tianjin Development Program for Innovation and Entrepreneurship (2018).

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