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.
Scherer et al. (2026) studied this question.