Live cells promote fibrillogenesis of biomimetic peptides through a heterogeneous secondary nucleation pathway

Our previous studies demonstrated that the binding-induced fibrillogenesis (BIF) biomimetic peptide lauric acid-FFVLK-HSDVHK (LAFH) underwent fibrillation in solution in the presence of Ca²⁺ through a heterogeneous secondary nucleation process. Herein, the specific mechanism governing the BIF process at the cellular level was studied. LAFH can specifically target the αvβ3 integrin of human umbilical vein endothelial cells (HUVECs) and form fibrous networks on the cell membrane. Cy5-labeled LAFH was incubated with HUVECs to study the fibrillogenesis kinetics of LAFH through its fluorescence signals. It was found that fluorescence of the cell membranes as well as fluorescence on a glass substrate, representing the LAFH nanofibers, could be used to study the BIF process. By fitting the time-dependent curves of fluorescence intensity on both cell surfaces and on glass substrates, it was determined that LAFH fibrillogenesis in the presence of HUVECs underwent heterogeneous secondary nucleation. We set up a fluorescence imaging assay and revealed a heterogeneous secondary nucleation mechanism for BIF of the biomimetic peptide LAFH at the cellular level, paving the way for precise regulation of BIF behavior in vitro and in vivo.