Efficient Cytoplasmic Delivery of Antisense Probes Assisted by Cyclized‐Peptide‐Mediated Photoinduced Endosomal Escape

Abstract

Intracellular delivery and endosomal release of antisense oligonucleotides remain a significant challenge in the development of gene‐targeted therapeutics. Previously, noncovalently cyclized TAT peptide (Cyc‐TAT), in which the final ring‐closing step is accomplished by hybridization of two short complementary γPNA segments, has been proven more efficient than its linear analogues at entering cells. As Cyc‐TAT also readily accommodates a binding site, that is, an overhanging γPNA sequence, for codelivery of functional nucleic acid probes into cells, we were able to demonstrate that the overhang‐Cyc‐TAT penetrated into A549 cells when carrying an anti‐telomerase γPNA that specifically reduced telomerase activity by over 97 %. Herein, we report that the cyclized TAT(FAM) can escape endosomes much more efficiently than the linear TAT(FAM) after LED illumination (490 nm). Based on this observation, the endosomal release of overhang‐Cyc‐TAT(FAM)/anti‐telomerase γPNA complex can be greatly enhanced by photoactivation, thus shortening cell treatment time from 60 to 3 h, while keeping the same high efficiency in inhibiting telomerase activity inside A549 cells.

Cellular uptake of 2‐aminopyridine‐modified peptide nucleic acids conjugated with cell‐penetrating peptides

“…[ 32 ] Following the methodology reported by Tan, Bruchez, and Armitage, after incubation with PNA‐Tat we irradiated the cells at 490 nm for 5 min to photoinduce endosomal escape. [ 49 ] However, we observed no changes to distribution pattern of PNA‐Tat before and after irradiation (Figure S17).…”

Targeting a Potential G-Quadruplex Forming Sequence Found in the West Nile Virus Genome by Complementary Gamma-Peptide Nucleic Acid Oligomers

“…43−45 Recently our lab has shown that a TAT peptide/γPNA chimera is capable of delivering an antisense γPNA probe (13-mer) and a variety of nucleic acidconjugated molecules into cells via hybridization to the γPNA overhang. 46,47 This approach should be amenable to delivering antiviral γPNAs into virus infected cells.…”

“…The use of polymeric nanoparticles comprised of biocompatible and degradable polymer poly­(lactic- co -glycolic acid) (PLGA) is an attractive cellular delivery method for γPNAs . This technique has been used to deliver γPNAs into cells as well as in a mouse model to induce gene correction. − Recently our lab has shown that a TAT peptide/γPNA chimera is capable of delivering an antisense γPNA probe (13-mer) and a variety of nucleic acid-conjugated molecules into cells via hybridization to the γPNA overhang. , This approach should be amenable to delivering antiviral γPNAs into virus infected cells.…”