Biophysical guided development of peptide-protein gels for 3D cell culture

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Bioengineered three-dimensional (3D) matrices hold considerable promise for regenerative medicine and cellular biology, and in particular for investigations of tumour growth. In this latter area, there is a need for improved three-dimensional (3D) cancer models to test responses to new treatments in a biologicallyrelevant, controlled environment. The 2D in-vitro cell models currently used fail to replicate many key features of tumours, including cell-matrix interactions and changes in matrix stiffness, and models that are more representative of the tumour microenvironment (TME) are needed to identify therapeutic strategiesmore quickly.Matrices based on self-assembling peptide-based materials have shown considerable recent promise, as they can be programmed to assemble into well-defined nanostructures representative of the extracellular matrix (ECM), and also optimized to enable tunability of key characteristics, such as mechanical properties. In this regard, recent research from the Mata group has demonstrated the potential of gels formed from peptide amphiphiles (PAs) co-assembled with proteins, to develop biologically relevant 3D cell cultures as models for ovarian1 and pancreatic cancer2.Despite their potential for a wide range of applications, the optimization/adaptation of such materials for defined disease models is however a lengthy process; there is a need to develop more rapid and rational analytical approaches to support this process. While it is relatively straightforward to test for biofunctionality, it is the analysis of other key characteristics such as matrix nanostructure and mechanical properties that present a significantly greater challenge, particularly in a hydrated environment. Biophysical techniques, such as atomic force microscopy (AFM), have shown considerable promise in this regard due theirunique ability to probe the structure and mechanical properties of matrix materials, with nanometre scale resolution3. This project will aim to develop a biophysical 'tool-box' to guide the development and more rapid identification/prediction of optimal peptide-protein gel properties for desired applications. The project will build on the expertise of Prof. Allen and Williams in the application of AFM and related biophysical approachesfor biomaterials analysis and development, in particular that related to peptide/protein self-assembly and aggregation. It will in addition build on very recent work initiated between Allen and Mata, demonstrating proof-of-concept AFM based measurements of Young's Modulus of his hydrogel materials.The 'tool box' will ultimately comprise one or more biophysical assays which will facilitate rapid identification of optimal conditions to provide the characteristics desired in a particular peptide-protein hydrogel system. To achieve this, initial studies (months 1-18) will focus on existing gel systems and will establish preliminary AFM based assays e.g. imaging studies to explore hydrogel structure and measurement/variation in material properties(stiffness, viscoelasticity). Comparison of data from the assays, with existing data and that obtained through bulk assays of material properties and performance in tissue culture, will allow initial evaluation of the AFM approaches to provide the required information (months 12-24). Initially the focus will be on well characterized peptide-protein hydrogels and the impact of simple experimental variables (e.g. media and buffers), before moving to more complex less well defined matricesincluding other biomolecules and/or cells. After the initial 'evaluation' phase, experiments will be performed to iteratively test and refine the biophysical approach(es) (months 18-36) and ultimately to enable evaluation of their ability to optimize and predict the performance of peptide-hydrogels for chosen applications (months 36-42).