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

Insulin aggregation poses a significant 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, which are known for disrupting protein aggregation via hydrogen bonding, electrostatic repulsion, and charge shielding. Among the screened peptides, Pep<inf>7</inf> (ERRVDRR) and Pep<inf>13</inf> (EKKVDKK) exhibited strong dose-dependent inhibition of aggregation. Thioflavin-T assays showed delayed fibrillation, with Pep<inf>7</inf> extending the lag time by 175% and reducing the aggregation rate by 67% at an insulin : Pep<inf>7</inf> 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<inf>7</inf>-containing insulin. Native PAGE and BCA assays indicated that Pep<inf>7</inf> retained 75 ± 3% monomeric insulin. DLS and TEM complemented the reduction in aggregate size, with TEM showing a diameter of 17.27 ± 3.53 nm for Pep<inf>7</inf>-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<inf>7</inf> to aggregation-prone regions of insulin (A1–A5, A18–A21, and B25–B29). Finally, MTT assays in HepG2 cells showed an enhanced viability of 78.55 ± 3.13% in peptide-containing samples. Collectively, these findings present Pep<inf>7</inf> and Pep<inf>13</inf> as promising peptide-based excipients for mitigating insulin aggregation and enhancing biopharmaceutical formulation stability, with potential utility as therapeutic agents for managing amyloid-associated proteinopathies.