Andrew R. Hirst

High‐Tech Applications of Self‐Assembling Supramolecular Nanostructured Gel‐Phase Materials: From Regenerative Medicine to Electronic Devices

It is likely that nanofabrication will underpin many technologies in the 21st century. Synthetic chemistry is a powerful approach to generate molecular structures that are capable of assembling into functional nanoscale architectures. There has been intense interest in self-assembling low-molecular-weight gelators, which has led to a general understanding of gelation based on the self-assembly of molecular-scale building blocks in terms of non-covalent interactions and packing parameters. The gelator molecules generate hierarchical, supramolecular structures that are macroscopically expressed in gel formation. Molecular modification can therefore control nanoscale assembly, a process that ultimately endows specific material function. The combination of supramolecular chemistry, materials science, and biomedicine allows application-based materials to be developed. Regenerative medicine and tissue engineering using molecular gels as nanostructured scaffolds for the regrowth of nerve cells has been demonstrated in vivo, and the prospect of using self-assembled fibers as one-dimensional conductors in gel materials has captured much interest in the field of nanoelectronics.

Biocatalytic induction of supramolecular order

Supramolecular gels, which demonstrate tunable functionalities, have attracted much interest in a range of areas, including healthcare, environmental protection and energy-related technologies. Preparing these materials in a reliable manner is challenging, with an increased level of kinetic defects observed at higher self-assembly rates. Here, by combining biocatalysis and molecular self-assembly, we have shown the ability to more quickly access higher-ordered structures. By simply increasing enzyme concentration, supramolecular order expressed at molecular, nano- and micro-levels is dramatically enhanced, and, importantly, the gelator concentrations remain identical. Amphiphile molecules were prepared by attaching an aromatic moiety to a dipeptide backbone capped with a methyl ester. Their self-assembly was induced by an enzyme that hydrolysed the ester. Different enzyme concentrations altered the catalytic activity and size of the enzyme clusters, affecting their mobility. This allowed structurally diverse materials that represent local minima in the free energy landscape to be accessed based on a single gelator structure. Supramolecular gels show promise in diverse areas, including healthcare and energy technologies, owing to tunable properties that arise directly from the organization of their building blocks. Researchers have now been able to control this behaviour by combining enzymatic catalysis with molecular self-assembly. Although it seems counter-intuitive, gels that assembled faster showed fewer defects.

Introducing chemical functionality in Fmoc-peptide gels for cell culture

Aromatic short peptide derivatives, i.e. peptides modified with aromatic groups such as 9-fluorenylmethoxycarbonyl (Fmoc), can self-assemble into self-supporting hydrogels. These hydrogels have some similarities to extracellular matrices due to their high hydration, relative stiffness and nanofibrous architecture. We previously demonstrated that Fmoc-diphenylalanine (Fmoc-F(2)) provides a suitable matrix for two-dimensional (2D) or three-dimensional (3D) culture of primary bovine chondrocytes. In this paper we investigate whether the introduction of chemical functionality, such as NH(2), COOH or OH, enhances compatibility with different cell types. A series of hydrogel compositions consisting of combinations of Fmoc-F(2) and n-protected Fmoc amino acids, lysine (K, with side chain R=(CH(2))(4)NH(2)), glutamic acid (D, with side chain R=CH(2)COOH), and serine (S, with side chain R=CH(2)OH) were studied. All compositions produced fibrous scaffolds with fibre diameters in the range of 32-65 nm as assessed by cryo-scanning electron microscopy and atomic force microscopy. Fourier transform infrared spectroscopy analysis suggested that peptide segments adopt a predominantly antiparallel beta-sheet conformation. Oscillatory rheology results show that all four hydrogels have mechanical profiles of soft viscoelastic materials with elastic moduli dependent on the chemical composition, ranging from 502 Pa (Fmoc-F(2)/D) to 21.2 KPa (Fmoc-F(2)). All gels supported the viability of bovine chondrocytes as assessed by a live-dead staining assay. Fmoc-F(2)/S and Fmoc-F(2)/D hydrogels in addition supported viability for human dermal fibroblasts (HDF) while Fmoc-F(2)/S hydrogel was the only gel type that supported viability for all three cell types tested. Fmoc-F(2)/S was therefore investigated further by studying cell proliferation, cytoskeletal organization and histological analysis in 2D culture. In addition, the Fmoc-F(2)/S gel was shown to support retention of cell morphology in 3D culture of bovine chondrocytes. These results demonstrate that introduction of chemical functionality into Fmoc-peptide scaffolds may provide gels with tunable chemical and mechanical properties for in vitro cell culture.

Enzyme responsive materials: design strategies and future developments

Enzyme responsive materials (ERMs) are a class of stimuli responsive materials with broad application potential in biological settings. This review highlights current and potential future design strategies for ERMs and provides an overview of the present state of the art in the area.

Self-assembly of two-component peptidic dendrimers: dendritic effects on gel-phase materials

The self-assembly of diaminododecane solubilised by different dendritic peptides, possessing increasing levels of dendritic branching, was investigated. The dendritic peptides were based on l-lysine building blocks and were of first, second and third generation, containing one, three and seven amino acid repeat units respectively. By applying these structures as potential gelator units, the dendritic effect on gelation was investigated. The degree of structuring was modulated, with the dendritic peptide controlling the aggregate morphology and the ability of the self-assembled state to manifest itself macroscopically as gelation. First generation gelator units (G1) did not induce macroscopic gelation with diaminododecane under any conditions, whilst those self-assemblies based on second (G2) and third (G3) generation branches did form gel-phase materials. Furthermore, gel-phase materials based on G2 exhibited optimum gelation behaviour compared to those based on G3(in terms of the thermal strength of the materials). Circular dichroism showed that the dendritic effect, programmed in at the molecular level, is directly related to the degree of chiral organisation within the self-assembled state. The dendritic generation of the peptide controls the pattern of amide-amide hydrogen bonding in terms of binding strength and alignment as determined using NMR methods. The mode of self-assembly can be qualitatively rationalised in terms of an attractive enthalpic interaction (i.e., amide-amide hydrogen bonding), a repulsive interaction (i.e., steric interactions between dendritic peptides) and an entropic term related to the hierarchical organisation of the gelator building blocks. It is argued that the balance between these factors determines the nature of the dendritic effect.

Supramolecular structures of enzyme clusters

The structural characterization of subtilisin mesoscale clusters, which were previously shown to induce supramolecular order in biocatalytic self-assembly of Fmoc–dipeptides, was carried out by synchrotron small-angle X-ray, dynamic, and static light scattering measurements. Subtilisin molecules self-assemble to form supramolecular structures in phosphate buffer solutions. Structural arrangement of subtilisin clusters at 55 °C was found to vary systematically with increasing enzyme concentration. Static light scattering measurements showed the cluster structure to be consistent with a fractal-like arrangement, with fractal dimension varying from 1.8 to 2.6 with increasing concentration for low to moderate enzyme concentrations. This was followed by a structural transition around the enzyme concentration of 0.5 mg mL–1 to more compact structures with significantly slower relaxation dynamics, as evidenced by dynamic light scattering measurements. These concentration-dependent supramolecular enzyme clusters provide tunable templates for biocatalytic self-assembly.