Inside Job: Methods for Delivering Proteins to the Interior of Mammalian Cells  Virginia J. Bruce, Brian R. McNaughton  Cell Chemical Biology  Volume 24, Issue 8, Pages 924-934 (August 2017) DOI: 10.1016/j.chembiol.2017.06.014 Copyright © 2017 Terms and Conditions

Figure 1 Common Approaches for Achieving Intracellular Delivery of Protein-Encoding Nucleic Acids (A) DNA/lipid-cation assemblies. (B) Viral vectors with encapsulated DNA. (C) Lipid bilayer deformation/physical insertion. Cell Chemical Biology 2017 24, 924-934DOI: (10.1016/j.chembiol.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 2 Cell-Selective Cell-Penetrating Peptides (A) Cell-selective delivery of protein cargo to cancer cells with cancer cell-activated cell-penetrating peptides (CPPs). Cell entry with a polycationic CPP is suppressed with a linked polyanionic sequence. However, when the polyanionic peptide is cleaved from the polycationic peptide, by an oncogenic matrix metalloprotease present in serum, the polycationic cell-penetrating peptide-protein cargo fusion enters neighboring (cancerous) cells. (B) Phage display selection for new cell-selective CPPs. A peptide phage display library is incubated with healthy cells and phage that do not penetrate these cells are enriched. Enriched phage are then incubated with diseased cells, and phage that penetrate these cells—by virtue of the phage-displayed peptide—are enriched. Cell Chemical Biology 2017 24, 924-934DOI: (10.1016/j.chembiol.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 3 Polycationic Resurfaced Proteins Can Potently Penetrate the Lipid Bilayer of Mammalian Cells Representative examples include (A) arginine grafted GFP, (B) supercharged GFP, and (C) polycationic resurfaced nanobodies. Mutated residues are highlighted as spheres (arginine, purple; lysine, blue). Nanobody complementary determining regions are highlighted in orange. Cell Chemical Biology 2017 24, 924-934DOI: (10.1016/j.chembiol.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 4 Cellular Delivery by Toxin-Derived Proteins B-type protein PA is recognized by a cell surface receptor (A). Following multimeric assembly of PA, (B) LFN (genetically fused to a cargo protein) is delivered, and recognized by the PA assembly. Following (C) endocytosis, PA (D) creates pores in the endosome membrane, leading to (E) translocation of LFN fusion through the PA assembly to enable cytosolic delivery of the cargo protein. Cell Chemical Biology 2017 24, 924-934DOI: (10.1016/j.chembiol.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 5 Phage- and Nanoparticle-Based Approaches to Protein Delivery (A) Engineered bacteriophage for delivery of exogenous proteins to prostate cancer cells. Phage are evolved to display a prostate cancer cell-selective CPP on the terminus of p3, and a biotin ligase receptor peptide on p9. Following biotinylation, phage are incubated with streptavidin (Sav) protein fusion, and incubated with cells. (B) Cationic capped gold nanoparticles are incubated with a polyanionic protein, such as β-galactosidase, to form a nanocomplex. Once formed this complex is taken up by mammalian cells, resulting in functional protein delivery. Cell Chemical Biology 2017 24, 924-934DOI: (10.1016/j.chembiol.2017.06.014) Copyright © 2017 Terms and Conditions

Figure 6 Delivery of CRISPR Protein-RNA Complexes to Mammalian Cells (A) CRISPR technology consists of the Cas9 endonuclease protein (light blue), complexed to a single guide RNA (sgRNA, orange); this engages genomic DNA (black) in a sequence-defined manner, based on the dictates of DNA:RNA duplex formation. Cas9 then makes a double-stranded break in genomic DNA, flanked by a protospacer adjacent motif (pam, white). The double-stranded break can be reconnected by non-homologous end joining or by homology-directed repair, using an exogenous piece of double-stranded DNA (dsDNA, purple). (B) The Cas9:sgRNA complex has a high theoretical negative charge. When pre-complexed with cationic lipids, delivery to the interior of mammalian cells is achieved. Cell Chemical Biology 2017 24, 924-934DOI: (10.1016/j.chembiol.2017.06.014) Copyright © 2017 Terms and Conditions