Calcium Ions Effectively Enhance the Effect of Antisense Peptide Nucleic Acids Conjugated to Cationic Tat and Oligoarginine Peptides

Abstract

Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in HeLa cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route for such peptides.

Photochemically enhanced cellular delivery of cell penetrating peptide‐PNA conjugates

“…4B). Finally, we compared the effect of co-treatment with PCI with that of the lysomotrophic agent chloroquine, which has been shown to enhance the antisense effect of PNA-Tat conjugates via endosome/lysozome release [5]. The results analyzed by luciferase activity as well as by RT-PCR splicing correction determination (Fig.…”

“…These peptides were all via continuous solid phase synthesis conjugated to a PNA oligomer targeting the cryptic splice site within the luciferase gene in HeLa pLuc705 cells. The nuclear antisense effect (as a positive luciferase activity readout) in this cellular system provides a quantitative measure that makes it possible to compare the relative nuclear uptake of the different PNA conjugates [5,19].…”

“…Thus the discovery and development of endosome-disruptive methods are of great interest and several have already been described such as Ca 2+ treatment [5], chloroquine treatment [6], sucrose treatment [7], and photochemical treatment [8]. Among these, photochemical treatment termed photochemical internalization (PCI) may be of particular interest because it has the potential for in vivo applications [9].…”

“…Although the mechanism of the PCI-mediated endosomal release is not yet fully understood [9], these promising results prompted us to test the effect of PCI on the antisense potency in cultured cells of a series of CPP conjugated PNAs which -in contrast to ''naked'' PNAs -are known to accumulate effectively in endosomes [2,5].…”

“…Recently, it has been found that cellular uptake of several cell penetrating peptide (CPP)-conjugates occurs predominantly via an endocytotic pathway [1,2] and that endosomal escape therefore is one of the major rate-limiting steps of CPP-mediated cellular delivery due to trapping of the CPP conjugates in the endosomal/lysosomal compartment [1][2][3][4][5]. Thus the discovery and development of endosome-disruptive methods are of great interest and several have already been described such as Ca 2+ treatment [5], chloroquine treatment [6], sucrose treatment [7], and photochemical treatment [8].…”