Elucidating the Impact of Payload Conjugation on the Cell-Penetrating Efficiency of the Endosomal Escape Peptide dfTAT: Implications for Future Designs for CPP-Based Delivery Systems

Cell-penetrating peptides (CPPs) are promising tools for the intracellular delivery of various biological payloads. However, the impact of payload conjugation on the cell-penetrating activity of CPPs is poorly understood. This study focused on dfTAT, a modified version of the HIV-TAT peptide with enhanced endosomal escape activity, to explore how different payloads affect its cell-penetrating activity. We systematically examined dfTAT conjugated with the SnoopTag/SnoopCatcher pair and found that while smaller payloads such as short peptides do not significantly impair dfTAT’s cell delivery activity, larger payloads markedly reduce both its endocytic uptake and endosomal escape efficiency. Our results highlight the role of the payload size and bulk in limiting CPP-mediated delivery. While further research is needed to understand the molecular underpinnings of these effects, our findings pave the way for developing more effective CPP-based delivery systems.