A Novel Photosensitizer for Light-Controlled Gene Silencing

Abstract

We here demonstrate for the first time that 5-carboxytetramethylrhodamine (TAMRA) covalently linked to nuclear localization signal (NLS)-conjugated peptide nucleic acids (PNAs) are photosensitizers (PSs) with the capacity to initiate photochemical damage to endocytic membranes, resulting in release of endocytosed material into cytosol. Our results show that TAMRA/PNA/NLS conjugates work as multifunctional molecules by offering cellular uptake, PNA-directed gene silencing, and the possibility for targeting in a light-controlled manner. In addition to PNA-directed gene silencing, we demonstrate that TAMRA/PNA/NLS molecules may function as a PS for light-controlled release of small interfering RNA molecules from the endocytic pathway when combined with an appropriate carrier. Using these strategies, we could silence the S100A4 gene at both protein and mRNA levels in a light-controlled manner, without any detectable reduction in cell viability. Our data demonstrate the possibility for light-controlled delivery of macromolecules entrapped within endocytic vesicles using multifunctional TAMRA/PNA/NLS molecules as PSs.

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

“…The structural DNA-mimic, peptide nucleic acid (PNA) is of interest in its own right as a third generation antisense agent, but has also been widely used as a model for studying cellular (and to a lesser extent in vivo ) delivery modalities 3 , 4 . It has previously been shown that cellular delivery of CPP-PNA conjugates can be enhanced by using PCI, through an auxiliary photosensitizer 5 , 21 – 23 or a covalently conjugated dye 24 , 25 . In the present work, we extend these studies by using fluorophore (Alexa Fluor 488 (AF488), Fluorescein, Alexa Fluor 555 (AF555) and Tetramethylrhodamine (TMR)) octaarginine PNA conjugates.…”

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

“…The acidification of late endosomes containing 1 was therefore expected to increase the aggregation state of 1 and further hinder its escape. Based on a small number of previous studies involving rhodamine-conjugated nanoparticles in cell cultures, − we speculated that “photochemical internalization” (PCI) could allow for the release of the vesicle-trapped TAMRA-dATP. Previous studies have demonstrated that irradiation of tetramethyl rhodamine at or near its excitation maximum results in the formation of singlet oxygen ( 1 O 2 ) and other reactive oxygen species (ROS), which oxidize and rupture the endosomal membrane. , Aside from potential applications in photodynamic therapy, we speculated that a subtoxic dose of light/ROS would release vesicle-trapped TAMRA-dATP into the cytosol and thereby facilitate its incorporation into DNA.…”

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

“…In an interesting study Bøe and co-workers [163] demonstrated light-induced endosomal release and gene silencing for a PNA oligonucleotide where a photosensitizing molecule was covalently attached to the oligonucleotide. The construct also included a positively charged nuclear localization signal peptide, thus constituting a multi-functional molecule offering cellular uptake, gene silencing, and the possibility for light-induced endosomal escape in one cargo.…”

“…Polycations can be used for effecting nucleic acid uptake by conferring a positive charge to the negatively charged nucleic acids so that a polycation/nucleic acid complex can be taken up after electrostatic interaction with the negatively charged cell surface. This is a general uptake mechanism that works very well in combination with PCI with many types of nucleic acids ranging from PNAs conjugated to positively charged amino acids [163,217] thorough poly-amino acid mediated delivery of plasmids and mRNA [159,166], to adenovirus coated with polycations [208]. To the extent that the employed polycations are not able to induce a "proton sponge" effect, such systems will often have very low gene delivery efficiency on their own, meaning that PCI can be used to literally turn delivery on, with enhancement factors up to >100 times being observed [85,190].…”

“…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.…”