Structural morphology of peptide nanofibrils dictates viral capture and cellular uptake in gene therapy applications

Peptide nanofibrils (PNFs) are gaining attention as promising transduction enhancers to improve viral vector delivery in ex vivo gene therapy applications. However, the influence of PNF structural morphology on viral capture and cellular interaction remains poorly understood. Here, we systematically compare two clinically relevant PNFs, D4, a short β-sheet-forming peptide, and Vectofusin-1, an α-helical peptide, focusing on their fibrillar architecture, viral particle binding, and interaction with host cells. Secondary structure analysis, molecular dynamics simulations and electron microscopy revealed that D4 forms loosely packed, β-sheet-rich aggregates, while Vectofusin-1 assembles into compact, α-helical structures. Both superstructures have a positive and hydrophobic surface which are key determinants for interaction with viral and plasma membranes. Upon exposure to virus-like particles (VLPs), D4 aggregates grew in size and density, while Vectofusin-1 formed more numerous, smaller aggregates. D4 bound VLPs with markedly higher density, yielding a uniform virion coating, in contrast to the lower and heterogeneous VLP association observed for Vectofusin-1. Notably, only D4 aggregates were actively engulfed by filopodia leading to active uptake via endocytosis mainly by macropinocytosis and subsequent degradation by lysosomes. In contrast, Vectofusin-1 binding to plasma membrane appeared more passive with minimal internalization. These distinct behaviors were maintained under transduction-like conditions, with D4 facilitating direct VLP contact with the plasma membrane and Vectofusin-1 forming extracellular networks. Our results reveal that PNF aggregate morphology critically determines viral and cellular interactions and suggest that D4 may offer superior efficacy and safety profiles for use in ex vivo gene therapies. Comparison of the transduction enhancer D4 and Vectofusin-1 from the molecular to macromolecular level and analysis of peptide nanofibril formation, aggregation behavior and interaction with virus-like particles (VLPs) and cells. Created in BioRender. Rauch, L. (2026) https://BioRender.com/wheubqi . • Morphology of peptide nanofibril (PNF) aggregates changes after virion binding. • PNF aggregate structure modulates virion binding capacity and their interaction with the cell membrane. • PNF structure and PNF aggregates morphology modulate cell membrane dynamics and uptake.