Conjugation to the Cell-Penetrating Peptide TAT Potentiates the Photodynamic Effect of Carboxytetramethylrhodamine

Abstract

BackgroundCell-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).Methodology/Principal FindingsWe 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.Conclusions/SignificanceTogether, 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.

Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide

“…This photon flux is approximately 3200 fold less than what has been reported for the irradiation of Fl-CPP trapped inside endosomes of live cells on an epifluorescence microscope (irradiance is high because of the focusing of light beam after 100× objective). For comparison purposes, 40 min irradiation with the described set-up is equivalent to 0.75 sec irradiation on a microscope, a time scale at which most endosomes are photolysed [7]. …”

“…However, while photosensitizers generally generate singlet oxygen in relatively high yields, the fluorophores used to label CPPs typically have very poor singlet oxygen quantum yields [14, 15]. Studies with membrane models such as the plasma membrane of red blood cells (RBCs) indicate that generation of ROS is indeed involved in the photolytic activity of Fl-CPPs [7, 16]. Yet, these studies also suggest that the CPP moiety acts in synergy with the singlet oxygen-generating fluorophores to disrupt membranes efficiently [16].…”

Functional peptides for siRNA delivery

“…Apoptosis and necrotic cell damage have been observed after laser excitation of R7-TPC and Tat-TMR [120, 127]. Cytoplasm membrane permeabilization and blebbing, and cell shrinkage were seen shortly after photo-illumination of TMR-TAT at 560 nm [127]. Another potential limitation of photochemically induced endosomolysis is the difficulty of in vivo light delivery.…”

TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells

“…These studies did not identify the cause of cell death but on the other hand suggested that cells could survive partial lysosomal disruption [13]. We have recently reported that TAT labeled with tetramethylrhodamine, TMR-TAT, also kills cells readily upon light irradiation during PCI experiments [14]. TMR-TAT efficiently escapes from endosomes on a millisecond time scale when using an epifluorescence microscope as light source.…”

“…TMR-TAT efficiently escapes from endosomes on a millisecond time scale when using an epifluorescence microscope as light source. The fluorescent peptide is also photolytic toward biological membranes such as the plasma membrane of red blood cells [14]. These effects involve the targeting of TMR by TAT to cellular membranes and the formation of singlet oxygen within membranes.…”