Improved Cellular Activity of Antisense Peptide Nucleic Acids by Conjugation to a Cationic Peptide-Lipid (CatLip) Domain

Abstract

Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.

Metal-containing peptide nucleic acid conjugates

“…New strategies for bioavailability such as conjugation to lipid domains 94 or oligophosphonates 95 were developed. Another novel and versatile idea is the use of metal complexes to increase cellular uptake.…”

Insights on chiral, backbone modified peptide nucleic acids: Properties and biological activity

“…For this reason, many researchers have focused their attention on developing new monomers to be used in the construction of second generation PNAs lacking the abovementioned issues. [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] Out of the above 1-3, water solubility is the least severe, with the exception of PNA's sequences rich in adenine and guanine. In addition, it should be noted that most of the cellular experiments are carried out at sub-micromolar or nanomolar.…”

“…67,68 The use of CPPs such as pTAT (RRRQRRKKR), transportan, 69 Antennapedia, 70 for example, allowed delivering PNA inside the cells as well as polyarginine or arginine rich peptides. 71,72 This approach has been extended to the use of cationic peptide-lipid conjugates 73 and to the newly discovered peptide carriers. 74 Nuclear localization signal peptide (NLS) has been shown to deliver the PNAs into the nucleus, but the cellular uptake is not enhanced as in other peptide cases.…”

Six-cell penetrating peptide-based fusion proteins for siRNA delivery

“…Except less efficiency in vivo (Li & Huang, 2007), the nonviral vector (compared to the viral vector) is a convenient delivery strategy with low toxicity and ease of large-scale production. Cell penetrating peptide (CPP) can transfer various cargos including protein (Choi et al, 2006;ElAndaloussi et al, 2007;Nakase et al, 2008), nuclear acid (Eguchi et al, 2001;Endoh & Ohtsuki, 2009), and the other drug vector (Koppelhus et al, 2008) into a range of cell types as well as the blood-brain barrier (Lindgren et al, 2000). However, the negative charge of siRNA may neutralize the positive charges of CPPs either by covalent attachment (Meade & Dowdy, 2007) or noncovalent conjugated (Veldhoen et al, 2006;Meade & Dowdy, 2008), and result to reduce efficiency of CPP.…”