Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

Atomic structures of naphthalene dipeptide micelles unravel mechanisms of assembly and gelation.

Peptide-based biopolymers have gained increasing attention due to their versatile applications. A naphthalene dipeptide (2NapFF) can form chirality-dependent tubular micelles, leading to supramolecular gels. The precise molecular arrangement within these micelles and the mechanism governing gelation have remained enigmatic. We determined, at near-atomic resolution, cryoelectron microscopy structures of the 2NapFF micelles LL-tube and LD-tube, generated by the stereoisomers (l,l)-2NapFF and (l,d)-2NapFF, respectively. The structures reveal that the fundamental packing of dipeptides is driven by the systematic π-π stacking of aromatic rings and that same-charge repulsion between the carbonyl groups is responsible for the stiffness of both tubes. The structural analysis elucidates how a single residue's altered chirality gives rise to markedly distinct tubular structures and sheds light on the mechanisms underlying the pH-dependent gelation of LL- and LD-tubes. The understanding of dipeptide packing and gelation mechanisms provides insights for the rational design of 2NapFF derivatives, enabling the modulation of micellar dimensions.

A Self-Assembling Flavin for Visible Photooxidation.

A new flavin-based gelator is reported which forms micellar structures at high pH and gels at low pH. This flavin can be used for the photooxidation of thiols under visible light, with the catalytic efficiency being linked to the self-assembled structures present.

Electrofabrication of large volume di- and tripeptide hydrogels via hydroquinone oxidation.

The fabrication of protected peptide-based hydrogels on electrode surfaces can be achieved by employing the electrochemical oxidation of hydroquinone to benzoquinone, liberating protons at the electrode-solution interface. The localised reduction in pH below the dipeptide gelator molecules pKa initiates the neutralisation, self-assembly and formation of self-supporting hydrogels exclusively at the electrode surface. Previous examples have been on a nanometre to millimetre scale, using deposition times ranging from seconds to minutes. However, the maximum size to which these materials can grow and their subsequent mechanical properties have not yet been investigated. Here, we report the fabrication of the largest reported di- and tri-peptide based hydrogels using this electrochemical method, employing deposition times of two to five hours. To overcome the oxidation of hydroquinone in air, the fabrication process was performed under an inert nitrogen atmosphere. We show that this approach can be used to form multilayer gels, with the mechanical properties of each layer determined by gelator composition. We also describe examples where gel-to-crystal transitions and syneresis occur within the material.

Controlling Syneresis of Hydrogels Using Organic Salts.

Supramolecular hydrogels can spontaneously undergo syneresis through fibre-fibre interactions and expel significant amounts of water upon aging. In this process, the hydrophobicity of fibres which regulates the 3D-rearrangement of the self-assembled structures during syneresis is important. Here, we show that we can control the hydrophobic microenvironment of gels by incorporating organic salts into the co-assembled gel fibres thereby enabling control of the macroscopic gel volume phase transition.

Exploiting and controlling gel-to-crystal transitions in multicomponent supramolecular gels.

Multicomponent supramolecular gels provide opportunities to form materials that are not accessible when using the single components alone. Different scenarios are possible when mixing multiple components, from complete co-assembly (mixing of the components within the self-assembled structures formed) to complete self-sorting such that each structure contains only one of the components. Most examples of multicomponent gels that currently exist form stable gels. Here, we show that this can be used to control the mechanical properties of the gels, but what is probably most exciting is that we show that we can use a magnetic field to control the shape of the crystals. The gelling component aligns in a magnetic field and so results in anisotropic crystals being formed.

Varying the hydrophobic spacer to influence multicomponent gelation.

Mixing low molecular weight gelators (LMWGs) shows promise as a means of preparing innovative materials with exciting properties. Here, we investigate the effect of increasing hydrophobic chain length on the properties of the resulting multicomponent systems which are capable of showing ambidextrous phase behaviour on pH perturbation.

Mechanical Characterization of Multilayered Hydrogels: A Rheological Study for 3D-Printed Systems.

We describe rheological protocols to study layered and three-dimensional (3D)-printed gels. Our methods allow us to measure the properties at different depths and determine the contribution of each layer to the resulting combined properties of the gels. We show that there are differences when using different measuring systems for rheological measurement, which directly affects the resulting properties being measured. These methods allow us to measure the gel properties after printing, rather than having to rely on the assumption that there is no change in properties from a preprinted gel. We show that the rheological properties of fluorenylmethoxycarbonyl-diphenylalanine (FmocFF) gels are heavily influenced by the printing process.

Programming Gels Over a Wide pH Range Using Multicomponent Systems.

Multicomponent hydrogels offer a tremendous opportunity for preparing useful and exciting materials that cannot be accessed using a single component. Here, we describe an unusual multi-component low-molecular weight gelling system that exhibits pH-responsive behavior involving cooperative hydrogen bonding between the components, allowing it to maintain a gel phase across a wide pH range. Unlike traditional acid-triggered gels, our system undergoes a change in the underlying molecular packing and maintains the ß-sheet structure both at acidic and basic pH. We further establish that autonomous programming between these two gel states is possible by an enzymatic reaction which allows us to prepare gels with improved mechanical properties.

Programming properties of transient hydrogels by an enzymatic reaction.

Supramolecular gels are usually stable in time as they are formed under thermodynamic equilibrium or at least in a deep well of a kinetically trapped state. However, artificial construction of kinetically controlled transient supramolecular gels is an interesting challenge. In these systems, usually a kinetically trapped transient aggregate is formed by active building blocks that leads to gelation; the gel then typically returns to the solution state. In this work, we show that such transient aggregation can occur by successive formation of two distinctly different kinetically controlled metastable states. Control over the first metastable state allows us to achieve significant control over the stability and properties of the second metastable state.

Using chirality to influence supramolecular gelation.

Most low molecular weight gelators are chiral, with racemic mixtures often unable to form gels. Here, we show an example where all enantiomers, diastereomers and racemates of a single functionalized dipeptide can form gels. At high pH, different self-assembled aggregates are formed and these directly template the structures formed in the gel. Hence, solutions and gels with different properties can be accessed simply by varying the chirality. This opens up new design rules for the field.

Using cavitation rheology to understand dipeptide-based low molecular weight gels.

The study of dipeptide-based hydrogels has been the focus of significant effort recently due to their potential for use in a variety of biomedical and biotechnological applications. It is essential to study the mechanical properties in order to fully characterise and understand this type of soft materials. In terms of mechanical properties, the linear elastic modulus is normally measured using traditional shear rheometry. This technique requires millilitre sample volumes, which can be difficult when only small amounts of gel are available, and can present difficulties when loading the sample into the machine. Here, we describe the use of cavitation rheology, an easy and efficient technique, to characterise the linear elastic modulus of a range of hydrogels. Unlike traditional shear rheometry, this technique can be used on hydrogels in their native environment, and small sample volumes are required. We describe our set-up and show how it can be used to probe and understand different types of gels. Gels can be formed by different triggers from the same gelator and this leads to different microstructures. We show that the data from the cavitational rheometer correlates with the underlying microstructure in the gels, which allows a greater degree of understanding of the gels than can be obtained from the bulk measurements.

Annealing multicomponent supramolecular gels.

Annealing is widely used as a means of changing the physical properties of a material. The rate of heating and cooling used in the annealing process controls the final properties. Annealing can be used as a means of driving towards the, or at least a, thermodynamic minimum. There is surprisingly little information on annealing kinetically-trapped supramolecular gels. Here, we show that annealing multicomponent gels can be used to prepare materials with tunable mechanical properties. We show that annealing in a two-component gel leads to a self-sorted network, which has significantly different mechanical properties to the as-prepared gels. Whilst the fibres are self-sorted, we show that the annealing of this system leads to significant change in the network level of assembly, and it is this that leads to the increase in storage modulus. We also show that it is possible to selectively anneal only a single component in the mixture.

P-Type Low-Molecular-Weight Hydrogelators.

As the use of low-molecular-weight gelators (LMWGs) as components in single and multicomponent systems for optoelectronic and solar cell applications increases, so does the need for more functional gelators. There are relatively few examples of p-type gelators that can be used in such systems. Here, the synthesis and characterization of three amino-acid-functionalized p-type gelators based on terthiophene, tetrathiafulvalene, and oligo(phenylenevinylene) are described. The cores of these molecules are already used as electron donors in optoelectronic applications. These newly designed molecules can gel water to form highly organized structures, which can be dried into thin films that show p-type behavior.

Using Aggregation-Induced Emission to Understand Dipeptide Gels.

We describe the formation of structured liquids and gels from a functionalised dipeptide based on tetraphenylethylene. Tetraphenylethylene is well-known to be able to undergo aggregation-induced emission. We use the emission data to understand the behaviour of the dipeptide in water under a variety of conditions. The dipeptide forms viscous solutions at high pH. Gels can be formed by a pH-trigger, but syneresis occurs. Addition of a calcium salt also leads to a gel with slight syneresis. Addition of sodium chloride leads to a self-supporting material, but this is not a true gel from the rheological perspective. From the emission data, we infer that there are limited structural changes on addition of sodium chloride or acid, but there are significant changes in molecular packing when the gel is formed by addition of a calcium salt.

Optimising low molecular weight hydrogels for automated 3D printing.

Hydrogels prepared from low molecular weight gelators (LMWGs) are formed as a result of hierarchical intermolecular interactions between gelators to form fibres, and then further interactions between the self-assembled fibres via physical entanglements, as well as potential branching points. These interactions can allow hydrogels to recover quickly after a high shear rate has been applied. There are currently limited design rules describing which types of morphology or rheological properties are required for a LMWG hydrogel to be used as an effective, printable gel. By preparing hydrogels with different types of fibrous network structures, we have been able to understand in more detail the morphological type which gives rise to a 3D-printable hydrogel using a range of techniques, including rheology, small angle scattering and microscopy.

Drying Affects the Fiber Network in Low Molecular Weight Hydrogels.

Low molecular weight gels are formed by the self-assembly of a suitable small molecule gelator into a three-dimensional network of fibrous structures. The gel properties are determined by the fiber structures, the number and type of cross-links and the distribution of the fibers and cross-links in space. Probing these structures and cross-links is difficult. Many reports rely on microscopy of dried gels (xerogels), where the solvent is removed prior to imaging. The assumption is made that this has little effect on the structures, but it is not clear that this assumption is always (or ever) valid. Here, we use small angle neutron scattering (SANS) to probe low molecular weight hydrogels formed by the self-assembly of dipeptides. We compare scattering data for wet and dried gels, as well as following the drying process. We show that the assumption that drying does not affect the network is not always correct.

Self-assembly, self-sorting, and electronic properties of a diketopyrrolopyrrole hydrogelator.

We show here the first example of a hydrogel formed from a symmetrically functionalised diketopyrrolopyrrole aromatic core. Gelation is triggered by change in pH. Not only does this gelator form aggregated, entangled structures at low pH, but it also forms worm-like micelles at high pH. The structures at high pH can be aligned using shear to provide conductive materials.