Mix & Gel—A New Strategy for the Development of Nanofibrous Cell Scaffolds Through Co-Assembly of Charge Complementary Binary Peptides

De novo self-assembling peptides have been widely exploited as bioinspired platform for the development of extracellular matrices that mimic natural mammalian cells microenvironmental niches.¹ Setting the molecular design rules for assembling peptides is crucial for the meticulous control of scaffold material properties over the length scale, to enable tailoring systems that can mimic the nanoarchitectural and micro environment for different cell types. We have recently adopted a minimalistic design approach for the development of ultrashort ionic-complementary constrained peptides (UICPs), rationally designed to assemble into thermodynamically stable β-sheet nanofibers with cell scaffolding potential.² While we have previously shown the importance of aromatic stacking for the assembly and packing of UICP nanofibres,² in this talk we will unravel the role played by charge interaction in tuning the molecular assembly, nanostructure and mechanical properties of charge co-complementary binary UICP mixtures.<br/>Our UICP binary mixtures are composed of a cationic peptide and an anionic counterpart, in which the charged residues are alternating around hydrophobic phenylglycine (Phg) and distributed in a pattern conferring a charge co-complementarity of the two sequences. Co-assembly into extended antiparallel β-sheet nanofibres, nanoribbons and twisted braids-like nanostructures was achieved upon mixing the charge co-complementary peptides and confirmed by FTIR, TEM and SAXS. Above critical gelation concentrations (~2-5 w/v%), these nanostructures spontaneously formed entangled networks and bulk hydrogels, as evidenced by the inverted vial test, oscillatory rheology and SEM. A full control over molecular co-assembly, size and morphology of nanostructures, as well as hydrogel stiffness, was possible via modulation of molar ratios of the binary components, the pH, and the overall peptide concentration. In contrast, mixtures of charge non-complementary sequences failed to co-assemble into any β-sheet-rich nanostructures, implying the importance of charge interactions for stabilizing nanostructure formation.<br/>Our suggested ‘Mix & Gel’ approach is specifically designed to facilitate re-suspension of cell pellets in one of the peptide solutions. Upon mixing with the ionic co-complementary partner peptide solution, spontaneous formation of homogenous 3D nanofibrous network occurs, encapsulating cells, with superior homogeneity compared to traditionally used cell-hydrogel encapsulation techniques. This technique tackles the common issue encountered when mixing cells with pre-formulated hydrogels such as impairment of cell viability upon repeated pipetting and difficulty of de-aggregating cells from pellets in the viscous hydrogel matrix. In addition, molar ratios of binary mixtures, pH and total peptide concentration can provide divergent hydrogels properties to suit the requirements of distinct cell phenotypes.<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 beam times (SM28806 and SM28287) to this project and to the staff, in particular, Andy Smith on beamline I22 and Charlotte J.C. Edwards-Gayle for their support with the SAXS experiments.<br/><b>References:</b><br/>[1] Ding X., Zhao H., <i>et al.</i>, 'Synthetic peptide hydrogels as 3D scaffolds for tissue engineering', <i>Advanced Drug Delivery Reviews.</i> <b>2020</b>; 160:78–104.<br/>[2] Wychowaniec J., Patel R., <i>et al.</i> 'Aromatic stacking facilitated self-assembly of ultra-short ionic complementary peptide sequence: β-sheet nanofibres with remarkable gelation and interfacial properties' <i>Biomacromolecules </i><b>2020</b>; 21: 2670-2680.