Fig. 2. Confocal imaging of cell penetration. BHK cells were treated for 1 h with DyLight 550 fluorescently labeled cargo proteins β-Gal (A), HRP (B) and myoglobin (C) (rendered as white in left panels, red in center and right panels), in either the absence or presence of TAT-CaM, washed and imaged live. Center images are optical sections set at a similar depth of the nucleus (NucBlue staining, white, center and right panels), as determined by position within the Z-stack. Orthogonal projections are shown at the right (boxed in red) and top (boxed in green) sides of each panel. Cytoplasmic compartments in live cells were visualized using CellTracker Green CMFDA dye (green in right panels). Comparison of TAT-CaM-treated versus untreated cells indicates that cargo proteins are entering the cell, and are localized primarily to the cytoplasm. Scale bars in all panels, 20 μm. Each experiment was replicated at least twice with the same results. 2474 ABSTRACTThe use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this ‘endosomal escape problem.’ Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

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