We report on how a dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with a particularly high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. Cytosolic delivery is achieved in most cells in a culture and only a relatively small amount of material remains trapped inside endosomes. Delivery does not require binding interactions between dfTAT and a protein, multiple molecules can be delivered at once, and delivery can be repeated. Remarkably, dfTAT-mediated delivery does not noticeably impact cell viability, proliferation, or gene expression. This new delivery strategy should be extremely useful for cell-based assays, cellular imaging applications, and the ex vivo manipulation of cells.

Endosomal entrapment is a severely limiting bottleneck in the delivery of biologics into cells. The compound dfTAT was recently found to circumvent this problem by mediating endosomal leakage efficiently and without toxicity. Herein, we report on the mechanism of endosomal escape of this cell-penetrating peptide. By modulating the trafficking of the peptide within the endocytic pathway, we identify late endosomes as the organelles rendered leaky by dfTAT. We establish that dfTAT binds bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes, and that the peptide causes the fusion and leakage of bilayers containing BMP. Together, these data identify late endosomes as desirable gateways for cell penetration and BMP as a cellular factor that can be exploited for the development of future delivery agents.

Edited by F. Peter GuengerichCell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from L-to D-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of L-to D-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the D-peptide, once in the endosome, is significantly more prone to escape than its L-counterpart. Moreover, the D-peptide is retained in the cytosol of cells for several days, whereas the L-peptide is degraded within hours. Notably, while the L-peptide is relatively innocuous to cells, the D-peptide exerts a prolonged antiproliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties. Cell-penetrating peptides (CPPs)2 have become important tools for the delivery of macromolecular cargoes inside cells (1-3). These delivery agents show promise in therapeutic applications and are useful reagents for cell biology assays (4 -6). For instance, CPPs (TAT, penetratin, etc.) are currently being tested in several preclinical and clinical trials (7,8). However, CPPs exposed to cells or serum are rapidly degraded, and this can consequently render these compounds less effective in vivo or in vitro (9 -13). To protect CPPs from degradation, a common strategy has been to employ D-amino acids instead of their L-amino acid counterparts. D-Peptides are protease-resistant, and this approach has been applied to CPPs such as TAT, R9, penetratin, hLF, pVEC, and sweet arrow peptide (10, 14 -17). In addition, the extended in vivo half-lives of D-peptides over L-peptides have contributed to the successful development of D-polyarginine CPPs as cancer contrast agents (18,19). How inversion of chirality impacts the multifaceted functions of CPPs, however, remains unclear.Several reports have indicated that cellular uptake of CPPs is independent of peptide backbone chirality (16,20). Uptake of the CPPs studied was thought to involve direct plasma membrane translocation. This is because uptake persisted at 4°C, a condition that typically abolishes endocytosis (16). In many cases, however, the penetration of CPPs into cells involves different routes of cellular entry (21). Instead of crossing the plasma membr...

Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density. We establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. Cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. Derivatives with multiple TAT branches induce the leakage of BMP-containing lipid bilayers, liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not cause leakage, induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in charge density of these branched structures leads to the emergence of lipid specific membrane-disrupting and cell-penetrating activities.

Endosomal entrapment is a common bottleneck in various macromolecular delivery approaches. Recently, the polycationic peptide dfTAT was identified as a reagent that induces the efficient leakage of late endosomes and, thereby, enhances the penetration of macromolecules into the cytosol of live human cells. To gain further insights into the features that lead to this activity, the role of peptide sequence was investigated. We establish that the leakage activity of dfTAT can be recapitulated by polyarginine analogs but not by polylysine counterparts. Efficiencies of peptide endocytic uptake increase linearly with the number of arginine residues present. In contrast, peptide cytosolic penetration displays a threshold behavior, indicating that a minimum number of arginines is required to induce endosomal escape. Increasing arginine content above this threshold further augments delivery efficiencies. Yet, it also leads to increasing the toxicity of the delivery agents. Together, these data reveal a relatively narrow arginine-content window for the design of optimally active endosomolytic agents.

Arginine-rich peptides can penetrate cells and consequently be used as delivery agents in various cellular applications. The activity of these reagents is often context-dependent, and the parameters that impact cell entry are not fully understood, giving rise to variability and limiting progress toward their usage. Herein, we report that the cytosolic penetration of linear polyarginine peptides is dependent on the oxidation state of the cell. In particular, we find that hypoxia and cellular antioxidants inhibit cell penetration. In contrast, oxidants promote cytosolic cell entry with an efficiency proportional to the level of reactive oxygen species generated within membranes. Moreover, an antibody that binds to oxidized lipids inhibits cell penetration, whereas extracellularly administered pure oxidized lipids enhance peptide transport into cells. Overall, these data indicate that oxidized lipids are capable of mediating the transport of polyarginine peptides across membranes. These data may also explain variability in cell-penetrating peptide performance in different experimental conditions. These new findings therefore provide new opportunities for the rational design of future cell-permeable compounds and for the optimization of delivery protocols.

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is however important in maintaining cellular homeostasis and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analog of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.