Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers.

Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI is an efficient vector for PNA delivery yielding significantly higher (up to 10-fold) antisense activity than an analogous PNA-octaarginine conjugate, even in the presence of chloroquine, which only slightly enhances the PEI-PNA activity. The PEI-PEG conjugates are preferred due to lower acute cellular toxicity. Finally, the method can be easily modified to allow for co-conjugation of other small molecules in a high-throughput screening assay that does not require a purification step.