We present a simple yet highly effective strategy to achieve a high degree of functional protein delivery into mammalian cells. Inefficient cellular uptake of protein-based drugs remains a major challenge for the biopharmaceutic industry. For example, antibody-drug therapy is restricted to targeting and blocking membrane-bound proteins, leaving a plethora of cancer-related intracellular targets untouched. Hence, we aim to develop efficient, effective, and biocompatible peptide-based protein delivery strategies.

One can achieve protein delivery through chemical conjugation with cell-penetrating peptides (CPPs). These protein-CPP conjugates are reported to enter cells directly via transduction through the cell membrane, albeit requiring high micromolar concentrations (>20 µM).^1,2 To address this, we developed the “CPP-additive technology” to facilitate greater direct transductions of protein-CPPs at low micromolar concentrations.³ CPP-additives work by accumulating on the cell membrane, resulting in the reduction of membrane tension to prime entry points of protein-CPP cargoes.

In this presentation, we introduce the second generation of CPP-additives containing a membrane-interacting hydrophobic motif. The hydrophobic CPP-additive enhanced the uptake via direct transduction of a model fluorescent protein compared to the original design. We show that the hydrophobic CPP-additive remains engaged on the cell membrane for a longer time (> 10 min) while creating loose membrane lipid packing defects at an earlier time point (~ 15 s). Finally, we demonstrate various applications for the delivery of biofunctional peptide- and protein cargoes facilitated by the hydrophobic CPP-additives.