Cell-penetrating peptides (CPPs) are sequences designed to enhance the cytosolic delivery of a range of cargoes. We are interested in delivering peptides containing the sequence DINNN, which are high-affinity inhibitors of the interaction between the SPRY domain-containing SOCS (suppressor of cytokine signaling) box (SPSB) proteins 1, 2 and 4 and inducible nitric oxide synthase (iNOS), and have potential as host-directed antibiotics. In order to assess the efficacy of CPPs in delivering these peptides to the cytosol of target cells, we have utilised the split luciferase endosomal escape quantitation (SLEEQ) assay, which measures the endosomal escape and cytosolic delivery of CPP conjugates. After applying correction factors to account for luminescence differences between peptides, the SLEEQ assay was used to evaluate CPP-peptide conjugates for their ability to enter a macrophage cell line. The CPPs TAT, (RW)and CPP9 all successfully enhanced cytosolic delivery of DINNN-containing peptides, but did so mainly by enhancing uptake into endosomes rather than by enhancing endosomal escape. Inclusion of the fluorophore sCy5 in the peptide conjugates further enhanced cytosolic uptake in general, although the impact varied significantly depending on the CPP used. Confocal imaging of the sCy5-containing peptide conjugates was consistent with the results of the SLEEQ assay, emphasising the risk of conflating CPP efficacy and dye effects when using fluorophore-based methods to study cell uptake. Our results for total cellular association and endosomal escape argue strongly for a mechanism of CPP uptake wherein interaction with the membrane and incorporation into endosomes is the key factor driving cytosolic delivery, rather than the efficiency of endosomal escape. While CPPs did enhance the cytosolic uptake of a DINNN-containing peptide, endosomal escape was found to be very inefficient, with none of the CPPs investigated here enhancing endosomal escape of the cargo. 4
