Evaluation of Cell-Penetrating Peptides (CPPs) as Vehicles for Intracellular Delivery of Antisense Peptide Nucleic Acid (PNA)

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Abstract
Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP−PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA−SS-Transportan-amide (ortho)−PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1−2 μM. (d-Arg)9−PNA showed optimal efficacy at 5 μM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat− and KFF−PNA conjugates showed significantly lower efficacy. The penetratin−, SynB3−. and NLS−PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA−SS-Transportan-acid (ortho) and (d-Arg)9−PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.