Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity

Abstract

Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates were investigated in terms of PCI assisted cellular activity. It is found that tetramethylrhodamine and Alexa Fluor 555 conjugated octaarginine PNA upon irradiation exhibit more than ten-fold increase in antisense activity in the HeLa pLuc705 luciferase splice correction assay. An analogous fluorescein conjugate did not show any significant enhancement due to photobleaching, and neither did an Alexa Fluor 488 conjugate. Using fluorescence microscopy a correlation between endosomal escape and antisense activity was demonstrated, and in parallel a correlation to localized formation of ROS assigned primarily to singlet oxygen was also observed. The results show that tetramethylrhodamine (and to lesser extent Alexa Fluor 555) conjugated octaarginine PNAs are as effectively delivered to the cytosol compartment by PCI as by chloroquine assisted delivery and also indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research

“…Folini and co-workers [92] used PCI with a naked peptide nucleic acid (PNA) to show that the PCI technology enhanced the biological activity of a PNA directed to the hTERT subunit of telomerase. Shiraishi and Nielsen [160] and Bøe and Hovig [161] further showed that PNAs conjugated to cationic peptides for the enhancement of cellular uptake were excellent substrates for PCI, with an PCI-induced enhancement of biological activity of > 100 times being achieved with some of the PNA constructs [160], also in accordance with other reports [162].…”

“…For many applications it could however seem advantageous to have the photosensitiser attached to the nucleic acid cargo, the delivery vehicle or both. Such systems have been developed both for oligonucleotides [162,163] and for plasmid [186] delivery, and has been shown to work well. While there are obvious advantages of having all components in one cargo, it however also makes the synthesis and formulation processes more complicated, and for some covalent systems it may also be a problem to achieve optimal doses of all components in the system at the same time.…”

Cooperative Cellular Uptake and Activity of Octaarginine Antisense Peptide Nucleic Acid (PNA) Conjugates

“…Therefore, high concentrations (micromolar) of CPP-conjugated PNA are typically required in order to obtain significant biological responses [14,15], making inefficient endosomal escape a major limitation encountered for CPP-PNA cellular delivery. Thus several enhancers such as chloroquine, calcium ions, or lipophilic photosensitizer have been identified for inducing more efficient endosomal release [21,22,23]. However, it has also been reported that endosomal leakage can be enhanced by increasing the concentration of some cationic CPPs (especially arginine-rich) inside the endosomes [24].…”

Codelivery of a cytotoxin and photosensitiserviaa liposomal nanocarrier: a novel strategy for light-triggered cytosolic release

“…We have however shown that the necessary amphiphilic and lysosomotropic properties can be engineered through the conjugation of otherwise highly hydrophobic photosensitisers to cationic cell-penetrating peptides (CPPs), to produce molecules that not only have the desired properties for PCI, but are superior to more classical tetrapyrrole-based derivatives 21,22. Other researchers have also adopted this principle of peptide-targeting with other photoactivatable dyes,23–26 and in this context, we recently demonstrated that CPP-conjugation can be a very attractive way of repurposing well-known photosensitisers that are clinically approved for PDT such as chlorin e 6 , which lack the appropriate physical properties for PCI 27. This could be easily achieved by the application of biorthogonal ligation chemistry for the attachment of the chosen photosensitiser to a readily available derivatised CPP, opening up the possibility of using other clinical PDT photosensitisers with the most attractive spectroscopic properties ( i.e.…”