Receptor-Specific Delivery of Peptide Nucleic Acids Conjugated to Three Sequentially Linked N-Acetyl Galactosamine Moieties into Hepatocytes.

Peptide nucleic acids (PNAs) are DNA analogs that bind with high affinity to DNA and RNA in a sequence-specific manner but have poor cell permeability, limiting use as therapeutic agents. The work described here is motivated by recent reports of efficient gene silencing specifically in hepatocytes by small interfering RNAs conjugated to triantennary N-acetyl galactosamine (GalNAc), the ligand recognized by the asialoglycoprotein receptor (ASGPR). PNAs conjugated to either triantennary GalNAc at the N-terminus (the branched architecture) or monomeric GalNAc moieties anchored at Cγ of three consecutive PNA monomers of N-(2-aminoethyl)glycine (aeg) scaffolds (the sequential architecture) were synthesized on the solid phase. These formed duplexes with complementary DNA and RNA as shown by UV and circular dichroism spectroscopy. The fluorescently labeled analogs of GalNAc-conjugated PNAs were internalized by HepG2 cells that express the ASGPR but were not taken up by HEK-293 cells that lack this receptor. The sequential conjugate was internalized about 13-fold more efficiently than the branched conjugate into HepG2 cells, as demonstrated by confocal microscopy. The results presented here highlight the potential significance of the architecture of GalNAc conjugation for efficient uptake by target liver cells and indicate that GalNAc-conjugated PNAs have possible therapeutic applications.