Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell‐penetrating synthetic model peptide

Abstract

In order to evaluate the ability of the cell-penetrating a-helical amphipathic model peptide KLALKLALKALK AALKLA-NH 2 (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5¢-GGAGCAGGAAAG-3¢ directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAPconjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three-and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10-and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.Keywords: cell-penetrating peptides; cellular uptake; PNApeptide conjugates.The wider application of peptide nucleic acids (PNAs) [1] as antisense agents appears to be limited mainly by poor cellular uptake [2,3]. Improved delivery into mammalian cells and enhanced antisense activity have been achieved after covalent coupling of PNAs to cell-penetrating peptides (CPPs), which are able to enter cells in a nonendocytic but as yet unknown mode [3][4][5][6][7][8]. The structural requirements for the delivery activity of peptides have been unclear until now. In order to contribute to an elucidation of structuredelivery activity relationships we have previously investigated the cellular uptake and biological activity of CPP-phosphorothioate oligonucleotide conjugates using the cell-penetrating amphipathic model peptide MAP (KLALKLALKALKAALKLA-NH 2 ) [9,10] as the lead compound [11]. The value of the results of this study was limited, however, by a high cell toxicity of the phosphorothioate oligonucleotide-peptide conjugates. Therefore, in the present study we evaluated the suitability of PNA to serve as the cargo molecule in MAP-based structuredelivery activity investigations. To this end we investigated cellular uptake and biological activity of a 12-mer peptide nucleic acid (5¢-GGAGCAGGAAAG-Lys-3¢; compound I; Table 1) complementary to bases 12-23 of the translated region of the nociceptin/orphanin FQ receptor, proven previously to be sensitive to antisense attacks [12,13], and of its conjugate with MAP (compound II; Table 1). For assessing the cellular uptake, we developed a protocol based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) providing absolute quantities of internalized PNA which was used supplementally with confocal laser scanning microscop...

Growth Factor- and Adhesion Protein-Like Components of Fetal Calf Serum Can Significantly Enhance the Intracellular Delivery of Peptide Nucleic Acids

“…1). In principle, this finding supports the reports claiming the ability of naked PNA to exert significant biological activity in mammalian cells also in the absence of delivery vectors (Oehlke et al, 2004;Turner et al, 2010). A 10 mm solution of scrambled PNA of identical base composition (PNA-2, Table) did not show any effect (not shown) implicating the observed splicing correction activity to be sequence specific (it was found to be true also after the below-described foregoing heating and also for Fluos-and PNA-2; Table 1).…”

“…In line with the latter reports, previously we found evidence for an extensive ability of PNA to cross mammalian plasma membranes (Oehlke et al, 2011) and to exert antisense activity in primary heart cells (Oehlke et al, 2004;Turner et al, 2010). Our results implied cell-type-dependent export by protection machineries rather than poor penetration into cells to be responsible for the mostly weak intracellular activity of PNA (Oehlke et al, 2011).…”

Cell Penetrating Peptides in the Delivery of Biopharmaceuticals

“…They have been used to mediate the delivery of PNAs and PMOs through covalent linkage [33]. The formation of efficient non-covalent complexes comprising CPPs and both charged and uncharged steric block oligonucleotides like 2'- O -methyl, LNA, PNA and charged PNA derivatives, has also been described [34]. …”

Conjugates of Oligonucleotides and Analogues with Cell Penetrating Peptides as Gene Silencing Agents

“…A conjugate of a 12-mer PNA with the amphipathic MAP peptide was taken up by neonatal rat cardiomyocytes 3-fold better than PNA alone and 8-fold better in CHO cells [106]. The conjugate augmented biological activity 5-fold directed against the nociceptin/orphanin FQ receptor compared to PNA alone, as a result of more rapid cellular uptake in combination with a lowered re-export and resistance to serum effects.…”