No / Peptides that self-assemble into nanostructures are of tremendous interest for biological, medical, photonic and nanotechnological applications. The enormous sequence space that is available from 20 amino acids probably harbours many interesting candidates, but it is currently not possible to predict supramolecular behaviour from sequence alone. Here, we demonstrate computational tools to screen for the aqueous self-assembly propensity in all of the 8,000 possible tripeptides and evaluate these by comparison with known examples. We applied filters to select for candidates that simultaneously optimize the apparently contradicting requirements of aggregation propensity and hydrophilicity, which resulted in a set of design rules for self-assembling sequences. A number of peptides were subsequently synthesized and characterized, including the first reported tripeptides that are able to form a hydrogel at neutral pH. These tools, which enable the peptide sequence space to be searched for supramolecular properties, enable minimalistic peptide nanotechnology to deliver on its promise.

In recent years, the degradation and subsequent loss of tissues is an issue that has affected people worldwide. Although there are treatments addressing the degradation of tissues, such treatments involve complicated and expensive procedures, where full tissue regeneration is not achieved. For these reasons, in recent years, tissue engineering has developed cutting-edge biomaterials capable of inducing effective tissue regeneration both under cellular or acellular conditions. Peptide hydrogels are versatile biomaterials composed of the basic components of life amino acids, which act as building blocks to form hierarchical structures, which subsequently go on to form well-defined scaffolds. Biomaterials have been widely used for the culture of mammalian cells, tissue engineering, regenerative medicine, drug delivery, etc. This is thanks to their capability of providing a three-dimensional architecture to cells, which mimics the natural architecture of the extracellular matrix (ECM). Peptide- based hydrogels can be easily functionalised with active biological cues, which can direct the cellular response. It has been shown that the ionic-complementary FEFEFKFK hydrogel, succeeded to support the culture of mammalian cells such as bovine chondrocytes. In this work, we used the same FEFEFKFK hydrogel to investigate the capability of this hydrogel to support the three-dimensional culture of both human osteoblasts (hOBs), and human mesenchymal stem cells (hMSCs) for bone regeneration applications. To achieve this goal, hOBs were cultured within both FEFEFKFK (non-functionalised) and RGD-FEFEFKFK (functionalised) gels. Then the suitability of the FEFEFKFK gels to induce cellular proliferation, synthesis of bone ECM and mineralisation was explored. In addition, taking advantage of the inherent plasticity of hMSCs, we also investigated the capability of the FEFEFKFK gel to foster the osteogenic differentiation of hMSCs, and subsequently to induce bone mineralisation in 3-D under osteogenic stimulation. Based on the results obtained in this work, the FEFEFKFK gel arises as a promising biomaterial for both bone and dental tissue regeneration applications.

Peptide-based biomaterials have gained much interest in various applications in drug delivery and tissue engineering in recent years, in large part due to their typically excellent biocompatibility and biodegradability. Composed of different amino acids, peptides can be designed with numerous sequences, providing flexibility and tunability in biomaterials. Peptides are easy to modify with small molecule drugs, inorganic components, and polymer chains to access multiple functions and tune properties relevant to biology and medicine. Stimuli-responsive peptide-based biomaterials can respond to environmental stimuli, such as light and ultrasound, in addition to local environmental factors, such as temperature, enzyme activity, and pH. Under environmental changes, these materials can be triggered to release therapeutic payloads, change conformations, or induce self-assembly in the target sites. In this work, I introduce the design, synthesis, and potential applications of several stimuli-responsive peptide-based biomaterials. The first half of this dissertation is based on enzyme-responsive, peptide-based biomaterials as extracellular matrix (ECM) mimics in tissue engineering. We synthesized linear and dendritic elastin-like peptides (ELPs) as crosslinkers and conjugated them with hyaluronic acid (HA) to form hydrogels. Trypsin was used as the enzyme trigger for cleaving the C-terminal lysine and to study how crosslinker topology affects enzymatic degradation. Hydrogels with dendritic ELPs degraded more slowly than linear ELPs, providing a novel strategy to tune the degradation rate of hydrogels as ECM mimics by the molecular design of crosslinker topology. Building on this peptide-polysaccharide platform for synthetic ECM design, we subsequently prepared hydrogels embedded with bioactive cryptic sites. These novel polymeric hydrogels mimicked native ECM cryptic sites by using depsipeptides that undergo an enzyme-triggered molecular rearrangement, "switching" from a non-functional epitope to a bioactive sequence. Mass spectrometry, 1H and 13C NMR spectroscopy, and fluorescence studies were applied to track structural changes in the peptide. SEM was used to image these polymer-peptide hybrid hydrogels. Finally, in vitro studies were conducted to evaluate cell interactions with the hydrogels. Switch peptide-modified alginate hydrogels showed increased cell adhesion upon induction of enzymatic activity, which provided a "gain of function" of the synthetic ECM. Critically, enzymes associated with the cells themselves could trigger the peptide switch and change in synthetic ECM behavior. With knowledge of stimuli-responsive peptide-based biomaterials applied in tissue engineering, I then studied how this system could be used in drug delivery by designing peptide-hydrogen sulfide (H2S) donor conjugates (PHDCs). H2S is a gasotransmitter that is produced endogenously, which has been explored in recent years with many potential therapeutical applications. We studied H2S release profiles in dual-enzyme-responsive PHDCs, with a further investigation into PHDC–Fe2+ complexes for potential tumor treatments via chemodynamic therapy. The PHDC–Fe2+ complexes were examined in a C6 glioma cell line, exhibiting an improved cell-killing effect compared with controls, by inducing toxic hydroxyl radical generation (•OH) via a Fenton reaction. To this end, we further discovered how side chains influence self-assembling nanostructures, H2S release profiles, and biological activities via three constitutionally isomeric PHDCs. Different morphologies and varied H2S release rates were observed, paving the way for tuning the properties of PHDCs by simple changes in molecular design. Finally, this dissertation discloses conclusions and future directions on stimuli-responsive peptide-based biomaterials using similar platforms with different designs in the drug delivery and tissue engineering fields. / Doctor of Philosophy / Peptides, short sequences of two or more amino acids linked by chemical bonds, are smaller versions of proteins. Forming naturally in nature, peptides are promising candidates in the design of biocompatible and biodegradable materials. To make these peptide-based materials "smart", certain sequences or functional groups are installed in the peptides, making them responsive to environmental changes, or stimuli. These external stimuli include light, ultrasound, temperature, enzyme activity, and pH changes. In this work, we have explored the design and synthesis of stimuli-responsive peptide-based biomaterials and their potential applications in tissue engineering and drug delivery. The first half of this dissertation focuses on the design and synthesis of two enzyme-responsive, peptide-based materials that function as extracellular matrix (ECM) mimics. The ECM is a three-dimensional microenvironment where cells reside, providing structural support and adhesive anchor points for cells. In the first system, we synthesized peptide-polysaccharide hydrogels with different peptide crosslinkers, comparing their enzymatic degradation performance to evaluate how peptide topology (architecture) influences degradation. A more branched topology led to a slower hydrogel degradation rate. To introduce biofunctionality into the ECM mimics, we embedded the second system with a "switchable" peptide sequence, which transformed from a non-functional peptide into a functional, bioactive epitope after being triggered by an enzyme. The functional peptide after the switch provided cell adhesion and increased cell spreading. The latter half of this dissertation explores the possibility of stimuli-responsive peptide-based biomaterials in drug delivery. We designed peptides that release hydrogen sulfide (H2S), a signaling gas is commonly known for its foul smell and toxicity, and studied the biological behaviors in cells. The peptide-H2S donor conjugates (PHDCs) were activated by the enzyme legumain, which cancer cells overproduce, leading to H2S release. With the combined treatment with Fe2+, the PHDC-Fe2+ system reduced cancer cell viability due to the high amount of hydroxyl radicals (•OH) generated by the Fenton reaction. This system may be a potential design platform for precise tumor treatments.

Hydrogen sulfide (H2S) is a gasotransmitter that is produced endogenously and freely permeates cell membranes. It plays important roles in many physiological pathways, and by regulating these pathways, it provides many therapeutic effects. For example, H2S dilates vascular vessels, promotes angiogenesis, and protects cells from oxidative stress. Due to its therapeutic effects, H2S has been used as a potential treatment for diseases like diabetes, ischemia-reperfusion injuries, lung diseases, ulcers and edemas, among others. To apply H2S for therapeutic applications, two challenges need to be addressed. The first challenge is the H2S donor, which not only provides H2S but must be stable enough to avoid side effects caused by overdose; and the second challenge is the delivery strategies, which transport the H2S to the target sites. A series of S-aroylthiooximes (SATOs), an H2S releasing compound, were synthesized and conjugated to peptide sequences to form H2S-releasing aromatic peptide amphiphile (APA) hydrogels. APAs formed nanofibers, which were stabilized by beta-sheets and aromatic stacking. The self-assembled structures were affected by the substituents on the aromatic rings of SATOs, leading to the formation of twisted nanofibers. After the addition of cysteine, H2S was released from the APAs with half-lives ranging from 13 min to 31 min. The electron-donating groups slowed down the H2S release rate, while the electron-withdrawing groups accelerated the release rate. Therefore, the release rates of H2S were controlled by electronic effects. When self-assembled structures were formed, the H2S release rate was slowed down even more, due to the difficulties in cysteine diffusion into the core of the structures. Antimicrobial effects were also discovered using the H2S releasing APA hydrogels. The H2S-releasing dipeptides S-FE and S-YE formed self-assembled twisted nanoribbons and nanotubes, respectively. The non H2S-releasing control oxime dipeptides C-FE and C-YE were also synthesized. The C-FE formed nanoribbons while the C-YE only showed non-specific aggregates. S-FE and S-YE released H2S with peaking times of about 41 and 39 min. Both the self-assembled structures and the release rates were affected by their packing differences. In vitro and ex vivo experiments with Staphylococcus aureus (Xen29), a commonly found bacterium on burn wounds, showed significant antimicrobial effects. APAs S-FE and C-FE eliminated Xen29 and inhibited the biofilm formation, while S-FE always showed better effects than C-FE. These antimicrobial H2S-releasing APA hydrogels provide a new approach to treat burn wound infections, and provide healing benefits due to the therapeutic effects of H2S. / Doctor of Philosophy / Hydrogen sulfide (H₂S) is a signaling gas that produced in our body. It regulates physiological pathways, and can be a potential treatment for diseases like diabetes, ischemia-reperfusion injuries, lung diseases, ulcers and edemas, among others. However, two issues need to be addressed before applying H₂S for disease treatments. The first issue is to choose an H₂S donor, which is stable enough to avoid side effects caused by overdose. The second issue is the delivery methods, which transport the H₂S to target sites. A series of S-aroylthiooximes (SATOs), an H₂S releasing compound, were synthesized and attached to peptide sequences to form H₂S-releasing self-assembled aromatic peptide amphiphile (APA) hydrogels. The APA hydrogels were found to be affected by the substituents on the SATO structures. For example, the H₂S released from APAs had halflives ranged from 13 min to 31 min, which were controlled by the substituents. When hydrogels were formed, the H₂S release was slowed down even more, due to the difficulties in cysteine diffusion into the SATO structures. The antimicrobial effects were also discovered using the H₂S releasing APA hydrogels. Two H₂S-releasing APA hydrogels, S-FE and S-YE, were formed. At the same time, two non H₂S-releasing oxime dipeptides, C-FE and C-YE, were also synthesized as controls. The H₂S-releasing peptides, S-FE and S-YE, released H₂S with peaking times of about 41 and 39 min, while no H₂S was released from C-FE and C-YE. The self-assembled structures and the release rates were affected by their structural differences. In vitro and ex vivo experiments with Staphylococcus aureus (Xen29), a commonly found bacterium on burn wound, showed significant antimicrobial effects. Both H₂S-releasing S-FE and non H₂S-releasing C-FE eliminated Xen29 and inhibited the biofilm formation, indicating the potential use of the designed peptides as antimicrobial treatment for wounds. The S-FE always showed better effects than C-FE, suggesting the benefit of H₂S during the elimination of bacteria. These antimicrobial H₂S-releasing APA hydrogels provide a new approach to treat burn wound infection and provide healing benefits due to the therapeutic effects of H₂S.

Progress in biomedicine requires the design of functional biomaterials, in particular, 3-dimensional (3D) scaffolds. Shear thinning, β-sheet based peptide hydrogels have attracted wide interest due to their potential use in tissue engineering and biomedical applications as 3D functional scaffolds. The emergence of carbon nanomaterials has also opened the door for the construction of increasingly functional hybrid hydrogels built from nanofibres and graphene-based materials using non-covalent physical interactions. The relationship between peptide molecular structure and the formed hydrogel is important for understanding the material response to shear. In particular, the physicochemical properties of peptide based biomaterials will affect the feasibility of injecting them during medical procedures. In the first part of this work, four peptides: FEFKFEFK (F8), FKFEFKFK (FK), KFEFKFEFK (KF8) and KFEFKFEFKK (KF8K) (F - phenylalanine, E - glutamic acid, K - lysine) were designed and used at identical charge to explore the effect of lysine rich β-sheet self-assembling sequences on the shear thinning behaviour and final properties of bulk hydrogels. By varying the peptide sequence design and concentration of the peptide, the tendency of the nanofibres formed to aggregate and the balance of nanofibre junction strength versus fibre cohesive strength could be explored. This allowed the existing theory of the shear thinning behaviour of this class of materials to be extended. The relationship between molecular structures of nanofibres forming the 3D network and the nano-filler is critical to understand in order to design tuneable and functional materials. In the next part of the work, three rationally designed β-sheet peptides, which form hydrogels: VEVKVEVK (V8), FEFKFEFK (F8) and FEFEFKFE (FE) (V - valine) and five graphene-based materials: graphene oxide (GO), reduced graphene oxide (rGO), three graphene-polymer hybrid flakes: GO with polydiallyldimethylammonium chloride (GO/PDADMAC), rGO with PDADMAC (rGO/PDADMAC) and rGO with polyvinylpyrrolidone (rGO/PVP) were used to form a selection of hybrid hydrogels. Graphene derivatives of the lateral flake sizes of 16.8 ± 10.1 µm were used. Various interactions between the graphene flakes and the peptides were observed that affected the overall mechanical properties of the hydrogels. Electrostatic interactions and pie-pie stacking, when phenylalanine residues are present, were shown to play a key role in determining the dispersion of graphene materials in the peptide hydrogels and stiffness of the hybrid materials. In particular, FE with reduced graphene oxide (rGO) and FE with rGO covered with polydiallyldimethylammonium chloride (PDADMAC) thin film formed double network-like hybrid hydrogels due to strong formation of peptide nanofibrillar bridges between adjacent rGO flakes. This corresponded to the 3- and 4-fold increase in the storage modulus (Gꞌ) of these hydrogels in comparison to controls. FE hydrogels with homogeneus dispersions of graphene oxide (GO) and reduced graphene oxide (rGO) are further shown to be suitable for 3D culture of human mesenchymal stem cells (hMSCs) with no cytotoxicity. These results focus attention on the importance of understanding interactions between the nano-filler and the nanofibrillar network in forming hybrid hydrogels with tuneable mechanical and biological properties, and demonstrates the possibility of using these materials as 3D cell culture scaffolds for biomedical purposes. Furthermore, graphene oxide (GO) itself is currently used in a number of processes of technological relevance such as wet spinning, injection moulding or inkjet printing to form graphene fibres, composites and printed conductors. Typically, such processes utilise well-aligned layered GO liquid crystal (LC) structures in aqueous dispersions. Flow and confinement encountered during processing affects the alignment and stability of this phase. In the final part of this work, the alignment of GOLCs of two lateral flake sizes (42.1 ± 29.4 µm and 15.5 ± 7.5 µm) were probed under a wide range of rotational shear flow conditions that overlap with the manufacturing processes defined by angular speeds from 0.08 to 8 rad.s-1 (and corresponding maximum shear rates from 0.1 s-1 to 100 s-1), in real-time, using shear induced polarized light imaging and small angle X-ray scattering, both coupled with an in-situ rheometer (Rheo-SIPLI and Rheo-SAXS, respectively). Under certain conditions, a unique pattern in Rheo-SIPLI: a Maltese cross combined with shear banding was observed. This phenomenon is unique to GO flakes of sufficiently large lateral size. The structure formed is attributed to a helical flow arising from a combination of shear flow and Taylor-vortex type flow, which is reinforced by a mathematical model. The orientations prescribed by this model are consistent with anomalous rheopecty oberved in Rheo-SIPLI and an anomolous scattering pattern in Rheo-SAXS. With the current trend towards producing ultra-large GO flakes, evidence that the flow behaviour changes from a Couette flow to a Taylor vortex flow was provided, which would lead to undesired, or alternatively, controllable alignment of GO flakes for a variety of applications, including aligned structures for biomedical purposes.