On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides.

The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.