Mirror image peptides have unique stability and immunogenic properties in mammals, making them attractive agents to investigate. Their properties inside cells have been mostly unexplored because biopolymers are difficult to transport across cellular membranes. Here, we used protective antigen (PA) from anthrax toxin to deliver mirror image polypeptide cargo into the cytosol of mammalian cells when conjugated to the C-terminus of the PA-binding domain of lethal factor, LFN. We found mirror image polypeptides and proteins were translocated as efficiently into cells as their L counterparts. Once in the cytosol, by the use of western blot, we found that d peptides at the C-terminus of LFN were able to achieve higher steady state concentrations when compared to the l-peptide conjugate. With this platform, we delivered a d-peptide MDM2 antagonist to disrupt the p53/MDM2 interaction in cancer cells. For the first time, we show the PA/LFN system is adaptable for the intracellular delivery of mirror image peptides and proteins.
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