Curd, Johnathan
(2022)
Customisable peptide hydrogels as user-defined biomimetic models of specific tissue microenvironments.
PhD thesis, University of Nottingham.
Abstract
The extracellular matrix (ECM) is comprised of a complex variety of biochemically distinct molecules including proteins, glycoproteins, proteoglycans, and polysaccharides. These provide not only the physical support for resident cells but also play a vital role in tissue homeostasis, by providing key biophysical and biochemical cues which regulate cell growth, survival, motility, and differentiation. However, current in vitro models typically fail the recapitulate the complexities of the native ECM, leading to a poor extrapolation of in vitro findings to in vivo relevance. This is a major contributing factor to the high attrition rate of novel drug candidates moving from the lab to the clinic. This has driven the recent interest in the development of more sophisticated 3D in vitro models which allow customisation of stiffness and composition to better reflect in vivo tissues.
However, currently there is not a single model platform which contains all the necessary features for a truly representative model. Naturally-derived biomaterials such as Matrigel® contain irrelevant animal-derived material, suffer from high batch-to-batch variability, and are poorly defined. All these features lead to difficulties in generating reproducible data and call into question whether these models truly reflect human tissues when they are composed of material of animal origin. Synthetic scaffolds and hydrogels have the advantages of being well defined, having limited batch variability, and containing no animal-derived material, but often lack complexity in terms of biochemistry. Whilst there are several methods available that allow for user-defined stiffness and porosity of these materials to better reflect the native architecture of target tissues, options for customisation with selected biomolecules to drive relevant cell behaviours are currently lacking.
The FEFEFKFK peptide hydrogel is a promising new candidate model platform that aims to address all the shortcomings of current in vitro models. Its stiffness can be controlled independent of its composition, allowing end users the ability to investigate the biological impact of matrix additions independently from tissue mechanics. Soluble matrix components selected to drive specific cell behaviours can also be encapsulated within the gel, allowing for a more faithful recreation of specific tissue microenvironments. However, at the beginning of this project, there existed only this single method for customisation of matrix composition. Potential loss of soluble material during cell culture, and the requirement for specific biomolecules to be covalently immobilised within the gel so that their correct in vivo behaviour is captured, necessitated the development of additional methods for customisation.
Therefore, it was the primary aim of this project to identify, optimise, and validate new methods for functionalising the peptide hydrogel with relevant biomolecules. Specifically, two methods for covalent immobilisation of matrix material were investigated: sortase-mediated and “click” chemistry-mediated functionalisation. Provided in this report is an initial proof of concept for these two methods and how immobilised material impacts the behaviour of encapsulated cells.
This work will allow for the creation of more complex and representative models of specific tissue microenvironments, without the need for animal-derived material. These models present researchers with the opportunity to study the underlying mechanisms of development and disease, or to better stratify novel drug candidates during preclinical screening, in an environment which produces more human-relevant and reproducible data. Compared to existing methods, the potential impact of this work will be to help reduce the high attrition rate of novel drug candidates moving from the lab to the clinic.
Actions (Archive Staff Only)
 |
Edit View |
Tools
Tools
![[thumbnail of Thesis]](https://eprints.nottingham.ac.uk/style/images/fileicons/application_pdf.png "Thesis")