Supplementary Material for: Effects of cell-adhesive ligand presentation on pentapeptide supramolecular assembly and gelation: Simulations and Experiments

The extracellular matrix (ECM) is a complex, hierarchical material containing structural and bioactive components. This complexity makes decoupling the effects of biomechanical properties and cell-matrix interactions difficult, especially when studying cellular processes in a 3D environment. Matrix mechanics and cell adhesion are both known regulators of specific cellular processes such as stem cell proliferation and differentiation. However, more information is required about how such variables impact various neural lineages that could, upon transplantation, therapeutically improve neural function after central nervous system (CNS) injury or disease. Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are one biomaterial approach to meet these goals, consisting of a family of peptide sequences that assemble into physical hydrogels in physiological media. In this study, we studied our previously reported supramolecularly-assembling RAPID hydrogels functionalized with the ECM-derived cell adhesive peptide ligands RGD, IKVAV, and YIGSR. Using molecular dynamics simulations and experimental rheology we demonstrated that these integrin-binding ligands at physiological concentrations (3-12 mM) did not impact assembly of the KYFIL peptide system. In simulations, molecular measures of assembly such as hydrogen bonding and pi-pi interactions appeared unaffected by cell-adhesion sequence or concentration. Visualizations of clustering and analysis of solvent accessible surface area (SASA) indicated that the integrin-binding domains remained exposed. KYFIL or AYFIL hydrogels containing 3 mM of integrin-binding domains resulted in mechanical properties consistent with their non-functionalized equivalents. This strategy of doping RAPID gels with cell-adhesion sequences allows for the precise tuning of peptide ligand concentration, independent of the rheological properties. The controllability of the RAPID hydrogel system provides an opportunity to investigate the effect of integrin-binding interactions on encapsulated neural cells to discern how hydrogel microenvironment impacts growth, maturation, or differentiation.

细胞外基质（extracellular matrix, ECM）是一类复杂的层级化材料，兼具结构组分与生物活性组分。这种复杂性使得解耦其生物力学性能与细胞-基质相互作用的效应颇具难度，在三维环境中研究细胞过程时尤为如此。已知基质力学与细胞黏附均为特定细胞过程的调控因子，例如干细胞增殖与分化。然而，关于此类变量如何影响各类神经细胞谱系——这些谱系经移植后可用于改善中枢神经系统（central nervous system, CNS）损伤或疾病后的神经功能——的相关信息仍有待补充。可注射快速组装五肽（Rapidly Assembling Pentapeptides for Injectable Delivery, RAPID）水凝胶便是契合此类研究目标的一种生物材料策略，其家族肽序列可在生理培养基中自组装形成物理水凝胶。本研究针对本课题组此前报道的超分子组装型RAPID水凝胶展开研究，该水凝胶经修饰搭载了ECM来源的细胞黏附肽配体RGD、IKVAV及YIGSR。通过分子动力学模拟与实验流变学分析，我们证实：生理浓度（3~12 mM）下的此类整合素结合配体，不会对KYFIL肽体系的组装过程产生影响。模拟结果显示，氢键与π-π相互作用等组装分子表征均不受细胞黏附序列及其浓度的干扰。聚类可视化分析与溶剂可及表面积（solvent accessible surface area, SASA）分析表明，整合素结合结构域始终处于暴露状态。搭载3 mM整合素结合结构域的KYFIL或AYFIL水凝胶，其力学性能与未功能化的对应水凝胶保持一致。这种向RAPID水凝胶中掺杂细胞黏附序列的策略，可在不改变流变学性能的前提下精准调控肽配体浓度。RAPID水凝胶体系的可控性为研究整合素结合相互作用对包封神经细胞的影响提供了契机，有助于阐明水凝胶微环境如何调控细胞生长、成熟或分化。