Dynamic reciprocity, the spatio-temporal bidirectional process between evolving partners in a functional system is not only found in nature, but also applies to supramolecularly assembling architectures. In this thesis, the focus was on the understanding of nature-inspired supramolecular self-assembly processes and mechanisms, with the aim to develop new assemblies for the use as functional biomaterials for medicine. The hierarchy, specificity and precision displayed by biological systems are the results of highly directional recognition interactions between the components of supramolecular structures. It is this control over the assembly and function that is interesting for chemistry. Understanding the interactions between non-covalently bonded molecules is crucial to the successful design and synthesis of self-assembling structures. This chemistry beyond the molecule is the field of supramolecular chemistry, and the development of supramolecular systems with precise control over structure and function with the same precision as displayed in nature can be seen as an ultimate goal. This objective can only be achieved if first the principles of self-organization and self-assembly processes exhibited by biological systems are fully understood. When these principles can be controlled, chemistry can develop macromolecular architectures that can interact with biological processes in a unique, dynamic reciprocal way. Natural systems form an unlimited source of inspiration for chemists and biologists to both understand and mimic. The fundamental mechanisms that play a role when multiple components, natural or synthetic, interact and form complex assemblies are dominated by the supramolecular chemistry in cyclic or linear polymerizations, multivalent clustering and molecular recognition. The composition and function of some particularly interesting natural multi-component assemblies, the extracellular matrix and lipid rafts, are reviewed. These systems display an impressive control over the spatio-temporal organization of multiple components. Examples of (partial) biomimicry of these systems, from fundamental insights toward functional biomedical applications form the starting point of this thesis. By mimicking natural complexity step-by-step, slowly but steadily we might start to better understand the lessons in multicomponent self-assembly provided by nature. Fundamental insights into the supramolecular polymerization mechanism of building blocks based on enzymes as the linking unit were obtained. The ribonuclease S-peptide and S-protein enzyme fragments were used to develop protein constructs. These fragments assemble via high-affinity molecular recognition and become enzymatically active when reconstituted. Supramolecular polymerization of the enzymatic building blocks was shown to obey the theory of macrocyclization under thermodynamic control. The results obtained contributed to the general understanding of supramolecular polymerization with biological building blocks and demonstrated design requirements for monomers if linear polymerization is desired. Another important fundamental phenomenon found in nature is multivalency; the strength of numbers. To explore the role of multivalent ligand presentation on binding affinity, the natural multivalent filamentous M13 phage was mimicked by a dendritic peptide construct. Phage display is widely used for the selection of target-specific peptide sequences and presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. The effects of valency, linker choice, and receptor density on binding affinity of the multivalent phage mimicking architecture were studied. When presented with a dense Streptavidin-coated surface, the synthetic phage mimic afforded up to an impressive 104-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The results obtained suggest that in vivo targeting schemes might best be served by a ligand selection under physiologically relevant receptor density surfaces. For the development of novel biomaterials with the aim to deliver growth factors and drugs, and mimic the natural extracellular matrix, a novel hydrogelator system was developed based on an ureido-pyrimidinone (UPy) end-capped poly(ethylene glycol) (PEG)-polymer. At physiological pH and acidic conditions, the UPy-hydrogelators form gel-like transient networks, though upon a pH increase their viscosity significantly drops and the system becomes fluid. To elucidate the hydrogel formation and the molecular phenomena playing a role at different pH, various analytical techniques were used to explore the molecular picture. Dynamic light scattering (DLS), atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) proved the existence of elongated UPy-fibers in the full pH range. Rheology demonstrated that the pH switching was reversible in terms of mechanical properties. With small-angle X-ray scattering (SAXS) experiments, the presence of molecular order in the gel system was demonstrated, which was sensitive to changes in pH, concentration and temperature. Translating the obtained fundamental knowledge into a medical application, the use of the pH-responsive behavior of the UPy-hydrogel system as injectable drug-delivery system was explored. With the state-of-the-art meter-long, narrow, medical NOGATM catheter system, the stimulation of cardiac regeneration after myocardial infarction by injection of growth-factor (GF) loaded UPy-hydrogel in pigs was investigated. Biocompatibility with cardiac progenitor cells and preservation of GF activity was shown, as well as retention of the injected payload after in situ gelation in porcine hearts. A significant reduction in infarct scar collagen content was achieved when the hydrogel was used to initiate endogenous cardiac repair by GF delivery. This simple intervention using a subtle pH switch to induce catheter-injectability of the hydrogel carrier meets the clinical demand of a minimally invasive delivery approach and shows potential for further follow up research. Tissue engineering uses the combination of cells with simulative scaffold materials to develop regenerative therapies. The scaffold is required to be a bioactive material that behaves both supportive, yet dynamic, and contains bioactive cues to stimulate the developing cell culture. Functionalization of supramolecular materials with guest compounds that display a complementary supramolecular recognition moiety is a common strategy to introduce functionality. To study the incorporation of (bioactive) guest compounds in the UPy-hydrogel fibers, a model system that can be monitored using fluorescence spectroscopy techniques was developed. Monovalent and bivalent UPy-functionalized fluorescein-labeled guest molecules were designed and their incorporation into the UPy-fibers was studied. Analysis of guest incorporation in the dilute state by microfluidics, as well as in the gel state, by fluorescence recovery after photobleaching, proved successful introduction of functionality into the fibers and provided insights into the fiber dynamics within the UPy-transient network. It was shown that the pH-induced disruption of the lateral UPy-fiber interactions in the material facilitated guest incorporation. The fundamental insights obtained from the measurements with the fluorescent model guest components were translated into the development of an artificial extracellular matrix for tissue engineering by replacing the fluorophore by bioactive peptide sequences that mimic the active sites in basement membrane (BM) proteins. The BM is tissue specific and each cell type requires ndividual optimization of composition of bioactive cues. The supramolecular mix-and-match approach of the UPy-hydrogel / UPy-guest system is proposed to be ideal to match with the specific cell-type needs on demand. In the early development stages of intestinal organoids, the BM is enriched in laminin-1. Therefore, a set of four laminin-1 derived peptides were synthesized and introduced into the UPy-hydrogel matrix. The organoid cell clusters showed expansion and budding in the first days of culturing compared to collapse in the non-functionalized hydrogel and gel with free-peptides. The development of a tailor-made synthetic BM for intestinal organoids was shown to be the first synthetic material able to induce expansion of these organoid cell clusters in vitro. Finally, all obtained knowledge on multivalency, UPy-hydrogel assembly, enzymatic building blocks and guest-host assembly was combined into the development of a synthetic supramolecular lipid raft mimic. Lipid rafts form the ultimate example of dynamic reciprocity and are characterized by microdomains in the outer leaflet of the cell’s plasma membrane with a spatio-temporal controlled existence, dependent on internal and external transient stimuli and interactions. Using the Ribonuclease S system, S-protein receptors, consisting of the UPy-polymer end-functionalized with two S-proteins, were placed on UPy-fibers. Dynamic clustering was induced by a multivalent S-peptide ligand that consisted of a hexavalent dendritic wedge, ligated to the S-peptide. By a time and concentration dependent increase in enzymatic activity, ligand-induced protein clustering was demonstrated. Full synthetic control over the rational design of a multi-component, dynamic, nature-inspired system was demonstrated, that helps to understand the mechanisms involved in multi-step non-covalent organization and to explore the boundaries of self-assembly to the fullest. The study of dynamic self-assembly, and resulting multi-step non-covalent synthesis procedures, is in its infancy. Exploring mechanisms found in biological processes and understanding the complex hierarchy of dynamic self-assembled structures in nature like lipid rafts, will lead to better guidelines for the design of bio-inspired mimics. In the last paragraphs of this thesis, an outline is provided on how this knowledge can become relevant for the development of supramolecular self-assembly into the multi-step non-covalent synthesis of novel structures. In the design of architectures for targeting purposes, the structural organization of the target should be carefully analyzed to prevent redundancy in ligand numbers. Similarly, when designing synthetic environments to direct tissue culturing, careful screening of the necessary active components in the natural cell environment prevents over-complication by addition of unnecessary signals. For future challenges concerning the UPy-system, both fundamentally and applied in medicine, further control over the dynamics of the transient network is required. When full control over both fiber and guest dynamics and stability can be obtained and controlled, UPy-based materials could be introduced in a wide range of targets in medicine. Cross-talk between scientists and medical doctors should bridge the gap between the laboratory and the clinic by identifying the needs for new biomedical developments. When common targets are identified, a joint effort is proposed to result in wonderful new discoveries, both for fundamental science and applied medicine.
|Qualification||Doctor of Philosophy|
|Award date||24 Sept 2012|
|Place of Publication||Eindhoven|
|Publication status||Published - 2012|