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.

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.

In the search of facile and efficient methods for PNA cellular delivery, we have tested a series of PNA conjugates based on (hetero) aromatic, lipophilic compounds such as 9-aminoacridine, benzimidazoles, carbazole, anthraquinone, porphyrine, psoralen, pyrene, and phenyl-bis-benzimidazole ("Hoechst"). These chemically modified PNAs were delivered to cultured pLuc705HeLa cells mediated by cationic liposomes (LipofectAMINE or LiofectAMINE2000), and their nuclear delivery was inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. PNAs modified with 9-aminoacridine, "Hoechst", or acetyl-"Hoechst" showed highest antisense activities (while unmodified PNA failed to show any significant antisense activity). In particular, bis-acridine-conjugated PNA showed nearly 60% splicing correction at 250 nM concentration in combination with LipofectAMINE2000. Interestingly, relative differences between the derivatives were observed when LipofectAMINE was used as compared to LipofectAMINE2000, but in general the latter yielded the higher antisense activity. The most active modifications of these PNA constructs were further tested for antisense down-regulation of luciferase in p53R cells in order to evaluate the cytoplasmic activity (uptake) of the PNAs. A dose-dependent down regulation of luciferase was demonstrated also in this system. The PNA conjugated to acetyl-Hoechst caused a reduction of luciferase activity to less than 40% of the control at a concentration of 1 muM. These results indicate that conjugation of (hetero) polyaromatic compounds to PNA can dramatically improve liposome-mediated cellular delivery both to cytoplasm as well as to the nucleus. However, no clear structure/activity relations are apparent from the present results, except that both 9-aminoacridine and "Hoechst" are also nucleic acid binding ligands.

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