13 research outputs found
Conjugation to the Cell-Penetrating Peptide TAT Potentiates the Photodynamic Effect of Carboxytetramethylrhodamine
Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents
Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges
Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed
Unlocking Endosomal Entrapment with Supercharged Arginine-Rich Peptides
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