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

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 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM 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.

Inhibition of Telomerase Activity Enhances Hyperthermia-Mediated Radiosensitization

“…After 24 h of PNA treatment, we found that 1 Amol/L hTR13-R9 produced the maximum inhibition of telomerase activity, whereas treatment concentrations >1 Amol/L decreased cell viability (Supplementary Fig. S1A; data not shown), in general agreement with another study using cell-penetrating peptide-PNA conjugates (36). We further compared the inhibition of telomerase activity at <1 Amol/L of PNA hTR13-R9, LNA, and GRN163L in cells treated for 24, 48, and 72 h ( Fig.…”

Penetratin-Mediated Transepithelial Insulin Permeation: Importance of Cationic Residues and pH for Complexation and Permeation

“…An increased number of arginine residues have previously been demonstrated to promote cellular uptake of CPPs (5,12), which most likely is a result of an improved hydrogen bonding between phospholipid headgroups in the cellular membrane and the CPP (7). However, earlier studies have also shown that an increased Arg content in a CPP negatively affects cell viability, possibly as a result of irreversible membrane perturbations mediated by the guanidinium groups (22,23). No decrease in viability was observed for PenArg in the concentrations employed in the present study (Fig.…”

The Biological Effect of an Antisense Oligonucleotide Depends on Its Route of Endocytosis and Trafficking

“…It should be noted that the uptake studies described here track the accumulation and retention of the fl uorophore; thus, it is possible that this measurement might not refl ect accumulation of the oligonucleotide itself if the labels were to be cleaved. However, it has been our experience Kang et al, 2008) and that of several other laboratories (Turner et al, 2005;Bendifallah et al, 2006;El-Andaloussi et al, 2006) that fl uorophore labels placed at the 3′ position of stable oligonucleotides provide reasonably accurate tracking of oligonucleotides in cell culture experiments such as those described here.…”