Integration of Leu-Asp-Val Cell Attachment Motif into Self-Assembling Peptide Sequences for Nanofibrillar Hydrogel Formation in Wound Healing

Abstract

Functionalizing peptide sequences with cell adhesion motifs enhances their cellular bioactivity. Numerous studies have focused on incorporating the Arg-Gly-Asp (RGD) motif into peptide hydrogels; however, the integration of other bioactive domains has yet to be comprehensively investigated. In this study, one of the essential fibronectin-derived cell-binding domains, Leu-Asp-Val (LDV), was integrated into the self-assembling peptide to obtain extracellular matrix (ECM)-mimetic nanofibrillar hydrogelators. IBP1A (NH₂-KLDVKLDVKLKV-CONH₂) and IBP1B (NH₂-KLDVKLDVKLDV-CONH₂) peptides were designed accordingly. These peptides self-assemble into hydrogels in phosphate-buffered saline (PBS) at pH 7.4 and deionized water at neutral pH with storage modulus values between ∼200 and ∼2000 Pa. Flow curves and the cyclic strain sweep data confirmed that the hydrogels have shear thinning, injectability, and self-healing properties. Flexible nanofibrillar morphology was observed in the TEM images. Nanofibril widths of IBP1A and IBP1B networks were measured as 8.2 ± 1.1 and 4.5 ± 0.8 nm, respectively. In vitro tests were also conducted to evaluate these peptides in wound healing applications. The IBP1A peptide with a +3 charge at neutral pH exhibited modest antibacterial activity against Gram (+) and Gram (−) bacteria. In vitro cell culture experiments show that the IBP1A and IBP1B hydrogels promoted the growth of fibroblast cells and glycosaminoglycan secretion compared with the KLDL12 control peptide, which does not contain the LDV motif. The designed hydrogels induced cell attachment within 72 h by altering the cell morphology similar to their natural 3D microenvironment, whereas cells exhibited spindle-like morphology on the KLDL12 hydrogel and tissue culture polystyrene (TCP). Moreover, IBP1B accelerated in vitro wound healing by facilitating fibroblast migration. These results suggest that these bioactive injectable peptide hydrogels have potential in wound healing and skin tissue regeneration.