Abstract
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.
Highlights
Cies are often sub-optimal, and in the absence of mechanistic insights, designing better tools often relies on a process of trials and errors
We establish that the cytosolic penetration of a CPP containing 13 arginine residues is correlated to the oxygen tension used during cell growth and to the presence of oxidants or antioxidants
Following the work of Futaki and co-workers [15, 18], Brock and co-workers [16], among others [17], we first tested the cytosolic penetration of linear polyarginine peptides containing an increasing number of arginine residues and labeled with The fluorophore 5(6)-carboxytetramethylrhodamine (TMR) by fluorescence microscopy
Summary
Cies are often sub-optimal, and in the absence of mechanistic insights, designing better tools often relies on a process of trials and errors. Cells were washed with heparin (1 mg/mL) solution and L-15 medium three times, followed by incubating with 1 M TMR-r13 for 10 min at 37 °C. The efficiency of cytosolic penetration was measured in MCH58 and HDF cells as the percentage of cells positive for nucleolar peptide staining and negative for SYTOX Blue staining.
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