Sequential synthesis of modular poly(ethylene glycol)—peptide hydrogels for nanoscale control over extracellular matrix features

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European Cells and Materials Vol. NN. Suppl. N, 20xx (page htu)

ISSN 1473-2262

Sequential synthesis of modular poly(ethylene glycol)—peptide hydrogels for nanoscale control over extracellular matrix features NJ Walters1, TT Yu2, OP Oommen3, J Hilborn3, S Miettinen1*, E Gentleman2* 1 BioMediTech, University of Tampere, Tampere, Finland. 2 Craniofacial Development & Stem Cell Biology, King’s College London, London, UK. 3 Polymer Chemistry, Department of Chemistry – Ångström Laboratory, Uppsala University, Uppsala, Sweden. INTRODUCTION: Stem cell behaviour and fate is influenced not only by mobile soluble signals, such as growth factors, but also by immobile characteristics of the extracellular matrix (ECM) 1. For example, on two-dimensional substrates, matrix stiffness 1, degradability 1,2 and cell adhesion ligand presentation 1,3 are all implicated in directing mesenchymal stem cell (MSC) differentiation via mechanotransductive processes. The shift over the last decade to three-dimensional (3D) cell culture models such as hydrogels has played an important role in elucidating the effect of stiffness under more in vivo-like conditions, better representing the dimensionality of most tissues. However, the effects of degradability and biomolecule presentation in 3D remain poorly understood. In the present research, modular poly(ethylene glycol) (PEG)–peptide hydrogels with an unprecedented degree of control over these factors have been developed. These materials allow each factor to be investigated either independently or in combination with others, minimising the effect of varying one factor from affecting other factors. METHODS: Hydrogels consisting of homofunctionalised four-arm PEG–nitrophenyl carbonate (PEG4NPC, synthesised as previously described 4) and PEG–vinyl sulfone (PEG4VS, JenKem Technology USA) – were sequentially cross-linked with custom-synthesised heterobifunctional peptides (Peptide Protein Research Ltd. UK) containing N-terminal Lys and C-terminal Cys residues. Peptides were linear (nonfunctionalised or containing a cleavable matrix metalloproteinase site) or T-shaped (containing branched adhesion ligands or growth factors). Stiffness was controlled by varying PEG molecular weight. Peptide mixtures of various ratios were first conjugated to PEG4NPC in phosphate buffered saline at pH 8.5. These conjugates were then crosslinked with PEG4VS under the same conditions to form hydrogels with varying properties. RESULTS: Hydrogels with various stiffness, degradability and biomolecule presentation were successfully synthesised via this approach. DISCUSSION & CONCLUSIONS: Synthetic PEG hydrogels are ideal for studying the effects of

ECM properties on stem cell differentiation, due to the bioinertness, non-immunogenicity and low protein adsorption of PEG. The use of multi-arm PEGs cross-linked via branched biomoleculepresenting peptides enables an unprecedented degree of control over individual or combined matrix properties at the nanoscale. Although diverse and highly innovative PEG hydrogels have been in use for over a decade 2, incorporated biomolecules such as cell adhesion ligands or growth factors have typically been attached to PEGs as “pendants”, preventing cross-link formation at those sites. This has a knock-on effect on other matrix properties, e.g. reduction of stiffness due to reduced crosslinking. The use of T-shaped peptide cross-linkers with biomolecules attached via branches largely overcomes this issue, enabling variation of one or multiple properties with minimal effects on others. Furthermore, the sequential nature of the reactions enables pre-formation of “clusters” – PEG4NPC with biomolecule-presenting peptides conjugated at desired ratios – which are then cross-linked via PEG4VS, forming a dendrimer-inspired hydrogel network. These materials are now being used to study the effects of matrix stiffness, degradability and biomolecule on differentiation of encapsulated MSC in 3D via gene and protein analyses.

Fig. 1: Sequential PEG–peptide gel assembly. Black/grey: PEG; blue: peptide; red: biomolecule. REFERENCES: 1 Walters NJ & Gentleman E. Acta Biomater. 2015;11:3–16. 2 Lutolf MP et al. Proc. Natl. Acad. Sci. USA. 2003;100:5413–8. 3 Arnold M et al. Chemphyschem 2004;5:383–8. 4 Walters NJ et al. Proceedings of Scandinavian Society for Biomaterials; poster presentation; 2016 Jun 1–3 Reykjavik, Iceland. ACKNOWLEDGEMENTS: Nothing to disclose. The authors wish to acknowledge OP Varghese for advice and Jane & Aatos Erkko Foundation, The Company of Biologists, Journal of Cell Science and Royal Society of Chemistry for funding. http://www.ecmjournal.org

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