Polymer-Protein Hybrid Thin Films through Co-Assembly: Optimisation, Characterisation, Functionalisation

Abstract

Stem cells have the remarkable ability to self-renew and differentiate into other cell types. The cell fate is regulated precisely by the cell’s microenvironment and the interaction of the cell with surrounding biomolecules. Cells sense their environment through a range of receptors, two of which are integrins and growth factor receptors. These receptors initiate cellular signalling pathways independently but also synergistically. While independent pathways are often well understood, there is a need for a more advanced understanding of the cross-talk between integrins and growth factor receptors. Many of these signalling pathways cannot be studied in vivo. Consequently, there has been tremendous progress in material systems that resemble aspects of the cells’ microenvironment in vitro. There is currently a lack of engineered biointerfaces to study synergistic signalling pathways between mechanotransduction and growth factor receptor signalling. Hence, this thesis aims to progress the development of a block copolymer-based platform that allows for the cell-mediated release of signalling molecules upon integrin binding. This would provide local release of biomolecules in close proximity to the integrin binding. The system comprises a polystyrene-block-poly(ethylene oxide) thin film containing proteins released upon exposure to an aqueous environment. Specifically, this thesis aims to quantify and control the release of protein from the block copolymer film. This thesis presents key milestones to contribute to the development of the proposed platform. The film fabrication parameters were optimised and resulted in largely defect-free films. Furthermore, the solvent-vapour annealing procedure was optimised, which resulted in the more reliable fabrication of cylindrical nanopatterns. Circular dichroism and an enzyme activity showed that the protein cargo retained its enzymatic activity in the solvent mixture used for the co-assembly of PS-b-PEO and protein cargo. The release rate into aqueous solutions from the co-assembled protein and polymer films was measured using detection of fluorescently labelled proteins and by an enzymatic assay. The results show a rapid release of incorporated protein. To hinder such fast release, the film needs to be capped. Work towards that goal in this thesis focussed on post-fabrication functionalisation of the thin film via copper‐catalysed azide/alkyne cycloaddition. The results presented in this thesis lay the foundation for the future use of the proposed system. However, particularly controlling the release from thin films in cell culture conditions needs further development to establish cell-mediated release of signalling molecules. Creating systems that allow for studying synergistic effects between mechanotransduction and growth factor signalling in vitro can further contribute to understanding stem cell behaviour in vivo. With extended knowledge and control about cellular processes, the use of stem cells in therapeutic applications becomes safer and more accessible to patients.