Effect of Charge on Molecular Self-Assembly, Nanoscopic and Bulk Material Properties of Ultra-Short Peptides

Bioinspired <i>de novo</i> self-assembling peptides have been widely used for the development of soft biomaterials for a wide variety of biomedical and pharmaceutical applications, such as cell scaffolding for tissue engineering¹, controlled and localised drug delivery², biosensing³, and many others. The meticulous control of peptide-based nanomaterial properties over the length scale, by molecular design, remains the main challenge for tailoring biomaterials properties to meet the application needs. In our group, we have recently adopted a minimalistic molecular engineering approach for the development of Ultrashort Ionic-complementary Constrained Peptides (UICPs), which were rationally designed to self-assemble into amphiphilic β-sheet nanofibers with unique hydrogelation properties and surface activity.⁴ We have previously demonstrated the crucial role played by aromatic stacking for the formation and thermodynamic stabilisation of UICP β-sheet structures. Herein, we will show how charge interactions can be manipulated for fine tuning molecular self-assembly, morphology and size of nanofibrous structures formation and viscoelasticity of UICP hydrogels.<br/><br/>A library of 18 peptide sequences (4-5 residues long) was developed to study the effect of the sequence net charge, charge density distribution, reversal of charge order and ionic self-complementarity on their propensity towards self-assembly and gelation. Interestingly, 12 of these peptides self-assembled into β-sheet nanofibrous structures forming hydrogels at pH 4.5-5, as confirmed by ATR-FTIR, SEM, TEM, SAXS and oscillatory rheology. Full control over β-sheet content (ranging from ~30-80%), fibre morphology (thin fibrils, thick straight fibre bundles, twisted helical nanofibres, flat nanoribbons and nanotubes) and sizes (~4-67 nm in diameter), as well as gelation (critical gelation concentrations ranging from <u>&lt;</u>7.5 to <u>&gt;</u>100 mM) and viscoelastic properties (storage moduli G’ ~0.1-100 KPa) was achieved by the careful positioning of both Glu and Lys residues at both C- and N-termini, in the sequence core and on both the hydrophilic and hydrophobic faces of the peptide chain. In essence, this design approach enabled/disabled lateral growth along the β-sheet ladder via electrostatic attraction (counter charge, anion-pi and cation-pi)/repulsion, hence controlling fibre thickness, morphology, entanglement, and the resulting viscoelasticity of the system. Our UICPs platform thus provides the flexibility in peptide molecular design for the manufacturing of soft biomaterials with versatile properties that can be in future tailored to the relevant biomedical application.<br/><b>Acknowledgements</b><br/>The authors would like to thank Rachel Armitage at De Montfort University for her assistance with SEM experiments and Natalie Allcock at University of Leicester for helping with TEM imaging. This work was funded by the Newton-Mosharafa fund awarded to A.K. and M.E. and the Egyptian Government missions’ sector scholarship awarded to M.S. The authors are also grateful to Diamond Light Source for awarding beamtimes (SM28806 and SM28287) to this project.<br/><b>References</b><br/>[1] Castillo-Diaz L., Elsawy M., <i>et al.</i> <i>Journal of Tissue Engineering</i> <b>2016;</b> 7: 1-15.<br/>[2] Elsawy M., Wychowaniec J., <i>et al.</i> <i>Biomacromolecules</i> <b>2022;</b> 23: 2624-2634.<br/>[3] Yousaf S., King P., <i>et al.</i> <i>Analytical Chemistry</i> <b>2019;</b> 91: 10016-10025.<br/>[4] Wychowaniec J., Patel R., <i>et al.</i> <i>Biomacromolecules </i><b>2020</b>; 21: 2670-2680.