Comparison of Receptor-Mediated Endocytosis and Its Application to Enhance DNA Transfection by TFAMoplex

Abstract

Receptor-targeting ligands can improve the specificity and cellular uptake of drugs and gene delivery systems. However, there is currently a lack of rapid, high-throughput assays to identify ligands that efficiently induce endocytosis. To address this gap, we developed a flow cytometric assay that selectively quantifies internalized fluorescence, directly converting endosomal uptake into an easily measurable signal across entire cell populations and avoiding the ambiguity and labor intensity of microscopy-based methods. Using this platform, we identified ligands that induce cellular uptake and applied them to the TFAMoplex, a modular, protein-based nonviral gene delivery system recently developed in our group. Coupling of heparin-binding epidermal growth factor, identified as an efficient internalizing ligand with the endocytosis assay, enhanced TFAMoplex uptake and transfection efficacy. These results support a generalizable strategy to rapidly screen uptake ligands, enabling the rational design of improved nonviral gene delivery systems.