Targeting HER2-positive cell lines by transmembrane peptide based surface-active ionic liquid nanoformulation

Abstract

Human epidermal growth factor receptor 2 (HER2/ErbB2) is a major oncogenic driver in many cancers, and its extracellular or intracellular domains are the main therapeutic targets. In contrast, the transmembrane domain (TMD), which also plays an important role in receptor dimerization and activation, remains comparatively underexplored as a potential therapeutic target. In this study, ErbB2 TMD-derived peptides were investigated as potential competitive inhibitors of receptor dimerization, while a nicotinium-based biamphiphilic surface-active ionic liquid (Nic BAIL) was developed as a vesicular carrier to address the poor aqueous compatibility of highly hydrophobic transmembrane peptides. Three ErbB2-TM peptide variants (v1-v3) were purified and characterized by LC-MS. Nic BAIL was synthesized and characterized by NMR, direct-infusion ESI-MS and differential scanning calorimetry (DSC). The Nic BAIL-v3 nanoformulation was prepared and characterized by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (Cryo-TEM), and its colloidal stability was evaluated under storage and dilution conditions. Planar bilayer electrophysiology was employed to investigate membrane activity, while cellular activity was evaluated in BT-474 and NCI-H23 cells, representing high and low ErbB2 expression, respectively. In addition, live-cell imaging was performed to observe morphological changes after treatment. Nic BAIL formed nanoscale vesicular assemblies, with a predominant nanoscale population maintained following formulation with v3 peptide, although the formulation exhibited time- and concentration-dependent changes in hydrodynamic size and dispersity. Planar bilayer electrophysiology showed that Nic BAIL-v3 increased ionic conductance and decreased electrical resistance across artificial DPhPC bilayers, indicating strongly altered membrane barrier properties. In cell viability assays, free v3 peptide produced similar concentration-dependent effects on BT-474 and NCI-H23 cells, while Nic BAIL-v3 produced an additional significant reduction in BT-474 viability compared to Nic BAIL alone. This enhancement was not observed in NCI-H23 cells. Live-cell imaging further demonstrated sustained cell-associated FAM-v3 fluorescence and progressive morphological deterioration without pronounced acute loss of membrane integrity. In this thesis, Nic BAIL was developed as a self-assembling membrane-active carrier for delivery of highly hydrophobic ErbB2-derived peptides and that Nic BAIL-v3 formulation exhibited a cell-line-dependent biological effect. But the intrinsic inhibitory activity of Nic BAIL complicates interpretation of observed cellular effects. Peptide loading, membrane localization, HER2-specific targeting, and direct interactions between v3 and HER2-TMD remain unconfirmed and require further investigation.