Speaker
Description
"Introduction
Short peptide amphiphiles have been widely reported as building blocks of supramolecular hydrogels for biomedical applications1,2, as they can copycat bioactive protein sequences. However, in the extracellular matrix (ECM), proteins are usually present as glycoproteins with different roles, e.g., storage depots of proteins and co-receptors. In this context, the use of self-assembling glycopeptide amphiphiles is gaining an increasing interest3 due to their ability to form supramolecular structures that mimic the ECM. In addition, the fact that they are maintained by non-covalent interactions (e.g., p-p or CH-p stacking) makes them inherently stimuli-responsive and dynamic systems.2 Here, we synthesised a short glycopeptide amphiphile, i.e., Fmoc-diphenylalanine-glucosamine-6-sulfate (Fmoc-FF-GlcN6S), and evaluated its ability to promote neural regeneration.
Methodology
Fmoc-FF-GlcN6S was synthesised by coupling Fmoc-FF and GlcN6S using DCC-NHS chemistry. Two methodologies were used to prepare the Fmoc-FF-GlcN6S hydrogels: 1) temperature switch (T method) – heating at 90 °C to dissolve the amphiphile followed by cooling to room temperature; and 2) solvent-switch (S method) – dissolving the amphiphile in DMSO followed by its dilution into water. The mechanical properties of the generated hydrogels were assessed by rheology, and their supramolecular structure (i.e., molecular packing/interactions and nanofiber morphology) was evaluated using CD, FTIR and AFM. The effect of the generated hydrogels on human adipose-derived stem cell (hASC) behaviour was evaluated by live/dead analysis, immunostaining and qPCR.
Results
Both methods formed hydrogels composed by entangled nanofibers. The stiffness of the hydrogels was influenced by the preparation method: gels generated by the T method were stiffer than the ones formed by the S method, G’ (T) = 2.4kPa > G’ (S) = 0.5kPa. Under both methods, the morphology of the nanofibers was similar (AFM). Interestingly, CD and FTIR analyses demonstrated that peptide glycosylation altered the secondary structure of the nanofibers from β-sheets (for Fmoc-FF) to α-helixes (for Fmoc-FF-GlcN6S).
hASCs cultured on Fmoc-FF-GlcN6S hydrogels showed distinct behaviour dependant on the preparation method: hASCs spread throughout the surface of the hydrogels prepared by the T method, while cell clusters were observed for hydrogels generated by the S method. qPCR and immunostaining showed that hASCs seeded on both types of hydrogels (i.e., T/S methods) overexpressed GFAP and Nestin on day three and MAP2 and βIII-tubulin on day nine of cell culture.
Conclusions
We demonstrate that Fmoc-FF-GlcN6S is able to self-assemble into biofunctional hydrogels, whose stiffness can be tuned altering the preparation method (i.e., S/T), as well as the concentration of the amphiphile. The Fmoc-FF-GlcN6S hydrogels induce hASC differentiation into neural lineages, being a good indication of their suitability for neural regeneration. In this context, the T method is a more adequate alternative as it does not use organic solvents and the gelation conditions (e.g., gelation timeframe) allows the encapsulation of cells.
Acknowledgements
We acknowledge the financial support from the EC (#668983-FORECAST and #964342-ECaBox) and FCT (PTDC/CTM-REF/0022/2020-OncoNeoTreat, PD/BD/135256/2017, COVID/BD/152018/2021 and CEECINST/00077/2018).
References
1.Ulijn, R. V. & Smith, A. M. Chem Soc Rev 37, 664-675, (2008).
2.Lampel, A. Chem 6, 1–15, (2020).
3.Brito, A. et al. Chem 7, 2943-2964, (2021)."
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