Surfactin-derived arginine-and lysine-rich peptides inhibit human insulin aggregation and prevent amyloid-induced cytotoxicity

Abstract

Insulin aggregation poses a major challenge in biopharmaceutical development and storage, compromising formulation stability, therapeutic efficacy, and patient safety through reduced bioavailability and immunogenic responses. Surfactin, a cyclic lipopeptide biosurfactant, has previously demonstrated aggregation-suppressing effects on insulin, providing a biocompatible alternative to conventional excipients like polysorbates, which are prone to hydrolysis and cytotoxicity. In this study, fourteen surfactin-inspired peptides were designed to mitigate insulin aggregation by replacing hydrophobic leucine residues with hydrophilic, positively charged arginine and lysine, amino acids known for disrupting protein aggregation via hydrogen bonding, electrostatic repulsion, and charge shielding. Among the screened peptides, Pep 7 (ERRVDRR) and Pep 13 (EKKVDKK) exhibited strong dose-dependent aggregation inhibition. Thioflavin-T assays showed delayed fibrillation, with Pep 7 extending the lag time by 75% and reducing aggregation rate by 67% at insulin:Pep 7 molar ratio of 1:5. Intrinsic tyrosine fluorescence and circular dichroism spectroscopy confirmed structural preservation, restoring 97% of Tyr fluorescence and near-native helical content in Pep 7 containing insulin. Native PAGE and BCA assays indicated Pep 7 retained 75 ± 3% monomeric insulin. DLS and TEM complemented the reductions in aggregate size, with TEM showing 17.27 ± 3.53 nm for Pep 7 -containing insulin. Isothermal titration calorimetry confirmed exothermic, spontaneous binding (ΔG = -16.97 kJ mol -1 ), supported by docking and 500 ns MD simulations that highlighted the preferential binding of Pep 7 to aggregation-prone HI regions (A1-A5, A18-A21, B25-B29). Finally, MTT assays in HepG2 cells depicted enhanced viability of 78.55 ± 3.13% in peptide-containing samples. Collectively, these findings present Pep 7 and Pep 13 as promising peptide-based excipients to mitigate insulin aggregation and enhance biopharmaceutical formulation stability, with potential utility as therapeutic agents for managing amyloid-associated proteinopathies.