GFP-complementation assay to detect functional CPP and protein delivery into living cells

The challenge for intracellular biologic drug delivery is achieving sufficient cytosolic uptake. A common solution is conjugating drugs to cell penetrating peptides (CPPs). Yet while many CPPs have been described, few CPP-delivered cargoes have entered the clinic1 with trials primarily dominated by just one CPP derived from the HIV transactivator protein (TAT)2,3. One reason so few of these therapeutics have made it to the clinic might be that many CPPs are generally inefficient at intracellular delivery and can remain largely trapped within endosomes4,5. Therefore, cargo proteins must normally be delivered at high concentrations exceeding 10 mg/kg to achieve biological efficacy6,7,8,9, but such high CPP concentrations become increasingly associated with cellular toxicity10. Differentiating between internalization and endosomal entrapment at therapeutically relevant concentrations is critical for discovering CPPs useful for research and for therapeutics. It is also critical to demonstrate that internalized peptide is available to mediate biological functions. Various methods have been described for detecting CPP uptake. But none can be reliably used for exclusive detection of cytosolic delivery, and hence biological availability, via a simple direct read-out. For example, assays using labeled CPPs can be affected by background signal from fluorescently-labeled CPPs trapped in endosomes, or from the labeling fluorophore11,12,13 or steroid14,15 itself over-enhancing membrane penetration and cytoplasmic uptake. Other options lack a direct signal readout, for example splice-correction or transcription-factor delivery assays that require processing of the signal or additional events for readout such as nuclear translocation and reporter-gene expression14,16,17,18,19. Alternative assays that rely on the redox potential of early endosomes to keep disulphide bonds oxidized20,21 find that premise may not always be the case, especially for artificial constructs22. All such factors complicate the interpretation of conventional CPP assays. This highlights the need for a diagnostic assay that specifically discriminates between cytoplasmic delivery and endosomal entrapment, is independent of the functional or differentiation state of the target cells, and also has a direct and easily-visualized readout with minimal background and sufficient sensitivity to detect CPP internalization at concentrations below toxic dosages. Our Split-complementation Endosomal Escape (SEE) assay visualizes cytosolic internalization of CPPs by fluorescence and thereby meets this need. The SEE platform is amenable to high-throughput processes and adaptable to different cell types (both transient and stable), making it a robust and sensitive diagnostic assay that specifically distinguishes functional uptake and cargo internalization in living cells.